Materials and methods for improved immunoglycoproteins

ABSTRACT

Immunoglycoproteins, including antibodies, with improved ADCC and altered glycosylation patterns are provided. Also provided are cell culturing methods and media for producing such immunoglycoproteins, and therapeutic uses of such immunoglycoproteins.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.12/082,497, filed Apr. 11, 2008, now U.S. Pat. No. 7,846,434, which is acontinuation-in-part of International Patent ApplicationPCT/US2007/082343 filed Oct. 24, 2007, which claims the benefit of priorU.S. provisional application no. 60/853,944 filed Oct. 24, 2006, all ofwhich are hereby incorporated by reference in their entirety.

FIELD OF THE INVENTION

The invention relates to immunoglycoproteins, including antibodies thathave improved properties, including antibody-dependent cell cytotoxicityand glycosylation patterns, cell culturing methods and media forproducing such immunoglycoproteins, and uses of such immunoglycoproteinsin treatment of disease.

BACKGROUND

Elimination of targeted cell populations with immunopharmaceuticals isan important therapeutic intervention in several indications. Themechanisms of action used by immunopharmaceuticals to effect suchelimination of targeted cells can include complement mediated cellularlysis, activation of apoptotic signaling pathways, blockade of signalingpathways required for survival, and antibody-dependent cellularcytotoxicity (ADCC), also referred to as Fc-dependent cellularcytotoxicity. ADCC is a potent mechanism that is believed to beimportant for the efficacy of many immunopharmaceuticals.

The mechanism for activation of ADCC involves binding of Fc receptors toimmunopharmaceutical molecules that are bound to the surface of thetarget cell. The binding of Fc receptors to immunopharmaceuticals can bemediated by domains within the constant region of immunoglobulins, suchas the CH2 and/or CH3 domains. Different types of constant regions maybind different Fc receptors. Examples include the binding of IgG1 Fcdomains to cognate Fc receptors CD16 (FcγRIII), CD32 (FcγRII-B1 and-B2), and CD64 (FcγRI), IgA Fc domains to the cognate Fc receptor CD89(FcαRI), and IgE domains to cognate Fc receptors FcεR1 and CD23.

Immunopharmaceutical compositions with enhanced Fc receptor binding mayexhibit greater potency in ADCC. Reported methods of achieving this withIgG Fc domains include the introduction of amino acid changes and themodification of carbohydrate structures. Modification of carbohydratestructures may be preferable as amino acid changes in the Fc domain mayenhance immunogenicity of a pharmaceutical composition. Forimmunoglobulin molecules it has been demonstrated that attachment ofN-linked carbohydrate (oligosaccharide) to Asn-297 of the CH2 domain iscritical for ADCC activity. Its removal enzymatically or throughmutation of the N-linked consensus site results in little to no ADCCactivity. Some studies have reported that the level of ADCC activity foran immunoglobulin molecule is also dependent on the structure of thecarbohydrate, but the actual carbohydrate moieties or structureresponsible for ADCC have not yet been elucidated. Still less is knownabout the optimal carbohydrate structure(s) for ADCC ofnon-immunoglobulin Fc fusion proteins.

In glycoproteins, carbohydrates may attach to the amide nitrogen atom inthe side chain of an asparagine in a tripeptide motif Asn-X-Thr/Ser.This type of glycosylation, termed N-linked glycosylation, commences inthe endoplasmic reticulum (ER) with the addition of multiplemonosaccharides to a dolichol phosphate to form a 14-residue branchedcarbohydrate complex. This carbohydrate complex is then transferred tothe protein by the oligosaccharyltransferase (OST) complex. Before theglycoprotein leaves the lumen of the ER, three glucose molecules areremoved from the 14-residue oligosaccharide. The enzymes ER glucosidaseI, ER glucosidase II and ER mannosidase are involved in ER processing.

Subsequently, the polypeptides are transported to the Golgi complex,where the N-linked sugar chains are modified in many different ways. Inthe cis and medial compartments of the Golgi complex, the original14-saccharide N-linked complex may be trimmed through removal of mannose(Man) residues and elongated through addition of N-acetylglucosamine(GlcNac) and/or fucose (Fuc) residues. The various forms of N-linkedcarbohydrates generally have in common a pentasaccharide core consistingof three mannose and two N-acetylglucosamine residues. Finally, in thetrans Golgi, other GlcNac residues can be added, followed by galactose(Gal) and a terminal sialic acid (Sial). Carbohydrate processing in theGolgi complex is called “terminal glycosylation” to distinguish it from“core glycosylation,” which takes place in the ER. The final complexcarbohydrate units can take on many forms and structures, some of whichhave two, three or four branches (termed biantennary, triantennary ortetraantennary). A number of enzymes are involved in Golgi processing,including Golgi mannosidases IA, IB and IC, GlcNAc-transferase I, Golgimannosidase II, GlcNAc-transferase II, Galactosyl transferase and Sialyltransferase.

One report has suggested that a crucial carbohydrate determinant ofFcγRIIIa receptor-mediated ADCC activity is the lack of analpha-1,6-fucose moiety added to the core N-linked structure (Shinkawaet al., J Biol Chem. 2003 Jan. 31; 278(5):3466-73; see also Shields etal., J Biol Chem. 2002 Jul. 26; 277(30):26733-40). The level of anotherglycoform, bisected N-linked carbohydrate, has also been proposed to becapable of imparting increased ADCC (Umana et al., Nat Biotechnol. 1999February; 17(2):176-80) but there is also contradictory evidence(Shinkawa et al., J Biol Chem. 2003 Jan. 31; 278(5):3466-73). Apotential solution to this contradictory evidence has been suggested bythe finding that increased GnTIII in host cells produces immunoglobulinnot only with increased bisected sugar but also lacking the core fucosemodification (Ferrara et al., Biotechnol Bioeng. 2006 Apr. 5;93(5):851-61). This agrees with suggestion that fucose alone has the keyrole in altering ADCC potency and the association with bisected sugarseen by others reflects a linkage in the two modifications in hostcells. However, another report in which in vitro treatment of Rituxanand Herceptin antibodies with GnTIII, to increase bisected sugar,resulted in increased ADCC suggests a direct effect of bisected sugar(Hodoniczky et al., Biotechnol. Prog., 2005 Nov.-Dec. 21 (6):1644-52).However, overexpression of Gnt III at very high levels may be toxic tothe cell (Umana et al., Biotechnol Prog. 1998 March-April;14(2):189-92).

Some proposed methods for producing immunoglobulins with lower fucosecontent have significant drawbacks for manufacture of abiopharmaceutical drug with an optimal ADCC activity for the therapeuticindication. For example, treatment of immunoglobulins with enzymes thatremove fucose residues (fucosidases) involves additional costlymanufacturing steps with potentially significant economic and drugconsistency risks. Molecular engineering of cell lines to knock-out keyenzymes involved in the synthesis of fucosylated glycoproteins requirespecial host strains and in current practice do not allow for “tunable”production of drug with varying ADCC potency to optimize efficacy andsafety for a therapeutic use. Generation of a comparison non-enhancedADCC product is expensive and time consuming. The treatment of celllines with RNAi or antisense molecules to knock down the level of thesekey enzymes may have unpredictable off-target effects and would becostly if not impractical to implement at manufacturing scale.

Thus, there continues to exist a need for advantageous methods ofpreparing immunopharmaceuticals with enhanced ADCC as well as for theimproved immunopharmaceuticals produced thereby for therapeutic uses.

SUMMARY OF THE INVENTION

The invention provides compositions of immunoglycoproteins with acharacteristic N-linked oligosaccharide content and improved properties,including effector functions such as ADCC. The invention also providesuses of such immunoglycoproteins and sterile compositions thereof intreatment of disease, culture media comprising such immunoglycoproteinsand/or host cells that produce them, and large scale cell culturemethods for improving the properties of such immunoglycoproteins. Theimmunoglycoprotein compositions of the invention can be characterized,for example, by glucose content or hexose content and/or fucose contentof the composition as a whole, depending on the number of N-linkedglycosylation sites for potential oligosaccharide linkage, or dependingon the number of N-linked oligosaccharides in the composition ofimmunoglycoprotein molecules. Alternatively, or in addition, theimmunoglycoprotein compositions of the invention can be characterized byglucose content, hexose content and/or fucose content of the N-linkedoligosaccharides. As yet another alternative, or in addition, theimmunoglycoproteins of the invention can be characterized by theglucose, hexose and/or fucose content of one or more major reducedimmunoglycoprotein species that represents a certain percentage of thecomposition.

The invention contemplates compositions comprising recombinantimmunoglycoprotein molecules comprising one or more N-linkedglycosylation sites for potential linkage of oligosaccharide. In oneaspect, the immunoglycoprotein molecules in the composition comprise anN-linked glycosylation site and have a glucose content characterized bya ratio of glucose molecules per said N-linked glycosylation site of atleast 1.2. In other exemplary compositions the ratio of glucosemolecules per said N-linked glycosylation site is at least about 1.4,1.5, 1.6, 1.8, 2, 2.2, 2.4, 2.6, or 2.8 or more. The composition ofimmunoglycoprotein molecules may also be characterized by a hexosecontent of at least about 9, 9.2, 9.4, 9.6, 9.8, or 10 or more hexosesper said N-linked glycosylation site, and/or a fucose content of lessthan about 1, 0.8, 0.6, or 0.4 or less, per said N-linked glycosylationsite.

In some embodiments, the immunoglycoprotein molecules of the compositioncomprise one or more CH2-derived domains, and at least one CH2-deriveddomain is characterized by a ratio of saccharide per N-linkedglycosylation site(s) of at least 1.2. In other exemplary compositionsat least one CH2-derived domain is characterized by a ratio of glucosemolecules per N-linked glycosylation site(s) of at least about 1.4, 1.5,1.6, 1.8, 2, 2.2, 2.4, 2.6, or 2.8 or more. The CH2-derived domain mayalso be characterized by a hexose content of at least about 9, 9.2, 9.4,9.6, 9.8, or 10 or more hexoses per N-linked glycosylation site, and/ora fucose content of less than about 1, 0.8, 0.6, or 0.4 or less, perN-linked glycosylation site.

In some embodiments, the immunoglycoprotein molecules comprise anN-linked glycosylation site, and a certain percentage, e.g., at leastabout 40%, of the N-linked oligosaccharides at said N-linkedglycosylation site have a glucose content of one to three glucosemolecules, e.g., 1, 2, or 3. In some embodiments, a certain percentage,e.g., at least about 40%, of the N-linked oligosaccharides at saidN-linked glycosylation site in the compositions of the invention have aglucose content of two to three glucose molecules. In exemplaryembodiments, such percentage is at least about 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90% or at least 95% or more. Thus, for example, insome embodiments, at least 40%, or at least 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90% or at least 95% or more of the N-linkedoligosaccharides at said N-linked glycosylation site have a glucosecontent of two glucose molecules. In some embodiments, at least 40%, orat least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or at least95% or more of said N-linked oligosaccharides of said immunoglycoproteinmolecules have a glucose content of three glucose molecules.

In some embodiments, the immunoglycoprotein molecules of the compositioncomprise one or more CH2-derived domains, and at least one CH2-deriveddomain is characterized by a certain percentage, e.g., at least about40%, of the N-linked oligosaccharides having a glucose content of one tothree glucose molecules, e.g., 1, 2, or 3. In some embodiments, acertain percentage, e.g., at least about 40%, of the N-linkedoligosaccharides of said CH2-derived domain have a glucose content oftwo to three glucose molecules. In exemplary embodiments, suchpercentage is at least about 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90% or at least 95% or more. Thus, for example, in someembodiments, at least 40%, or at least 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90% or at least 95% or more of said N-linkedoligosaccharides of said CH2-derived domain are linked to two glucosemolecules. In some embodiments, at least 40%, or at least 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90% or at least 95% or more of saidN-linked oligosaccharides of said CH2-derived domain are linked to threeglucose molecules.

In some embodiments, a certain percentage, e.g., at least about 40%, ofthe N-linked oligosaccharides at said a N-linked glycosylation site inthe compositions of the invention have a hexose content of at least tenhexoses. In some embodiments, a certain percentage, e.g., at least about40%, of the N-linked oligosaccharides at said N-linked glycosylationsite in the compositions of the invention are characterized by a hexosecontent of ten hexoses. In some embodiments, such percentage is at leastabout 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or at least 95%or more of said N-linked oligosaccharides of said immunoglycoproteinmolecules.

In some embodiments, the immunoglycoprotein molecules of the compositioncomprise one or more CH2-derived domains, and at least one CH2-deriveddomain is characterized by a certain percentage, e.g., at least about40%, of the N-linked oligosaccharides of said CH2-derived domain have ahexose content of at least ten hexoses. In some embodiments, a certainpercentage, e.g., at least about 40%, of the N-linked oligosaccharidesof the CH2-derived domains are characterized by a hexose content of tenhexoses. In some embodiments, such percentage is at least about 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or at least 95% or more ofsaid N-linked oligosaccharides of said CH2-derived domains.

Any of the preceding compositions of recombinant immunoglycoproteinmolecules may exhibit at least about 2-fold, 3-fold, 4-fold or 5-foldhigher ADCC or CD16-binding compared to control immunoglycoproteinmolecules of the same encoded amino acid sequence produced in CHO-K1cells in the absence of carbohydrate modifier. In other embodiments, theimmunoglycoprotein molecules exhibit at least about 6-fold, about7-fold, about 8-fold, about 9-fold, about 10-fold, about 20-fold, about30-fold, about 40-fold, about 50-fold, about 60-fold, about 70-fold,about 80-fold, about 90-fold, about 100-fold or more higher ADCC orCD16-binding compared to control immunoglycoprotein molecules of thesame encoded amino acid sequence produced in CHO-K1 cells in the absenceof carbohydrate modifier. Improved binding to the high affinity, lowaffinity, or advantageously both types of CD16 alleles (V158 highaffinity and F158 low affinity) is observed.

Any of the preceding compositions of recombinant immunoglycoproteinmolecules may further be characterized by a fucose content wherein atleast about 60% of the N-linked oligosaccharides, e.g. N-linkedoligosaccharides in the CH2-derived domains, contain no fucose. Inrelated embodiments, the percentage of N-linked oligosaccharides, e.g.N-linked oligosaccharides in the CH2-derived domains, that contain nofucose is at least about 65%, about 70%, about 75%, about 80%, about85%, about 90%, about 95% or more.

In some embodiments of the invention, such immunoglycoprotein moleculescomprising a N-linked glycosylation site in the compositions of theinvention exhibit more than one of the foregoing properties, forexample, (1) glucose content (e.g., of the CH2-derived domain)characterized by a ratio of glucose molecules per said N-linkedglycosylation site of at least 1.2, (2) at least 40% of the N-linkedoligosaccharides (e.g., of the CH2-derived domain) at said N-linkedglycosylation site have a glucose content of two to three glucosemolecules, (3) at least 60% of the N-linked oligosaccharides (e.g., ofthe CH2-derived domain) at said N-linked glysosylation site have ahexose content of ten hexose molecules, (4) at least 5-fold higher ADCCcompared to control immunoglycoprotein molecules of the same encodedamino acid sequence produced in CHO-K1 cells in the absence ofcarbohydrate modifier, and (5) at least 60% of the N-linkedoligosaccharides (e.g., of the CH2-derived domain) at said N-linkedglycosylation site contain no fucose.

Exemplary immunoglycoproteins of the invention include immunoglobulinsand small, modular immunopharmaceutical (SMIP™) products. Such bindingmolecules advantageously retain substantially the same properties ofbinding to target and resulting direct biological activity, but exhibitimproved effector-mediated functions.

In some embodiments of the invention, the recombinant immunoglycoproteinmolecules, in a reduced state, have a single N-linked glycosylationsite. In other embodiments, the recombinant immunoglycoprotein moleculeshave two, three, four or more N-linked glycosylation sites. In someembodiments of the invention, the immunoglycoprotein molecules aremonovalent, while in other embodiments, they are multivalent, e.g.,bivalent, trivalent, tetravalent or higher multivalencies. In someembodiments, the immunoglycoprotein molecules are monomeric, while inother embodiments, they are multimeric, e.g., dimeric, trimeric,tetrameric, or higher multimeric. In some embodiments, theimmunoglycoprotein molecules are monospecific for a target, while inother embodiments, they are multispecific, e.g., bispecific,trispecific, or higher multispecific in antigen-binding. It iscontemplated that in some embodiments, for example, within a multimericmolecule, the N-linked oligosaccharide (e.g., of the CH2-derived domain)on one monomer of the multimer may be different from the N-linkedoligosaccharide on another monomer within the same multimer. Forexample, within an immunoglycoprotein molecule, one N-linkedoligosaccharide (e.g., of the CH2-derived domain) may have two glucoseslinked thereto, while the other N-linked oligosaccharide may have noglucoses linked thereto.

In another aspect, a method of the invention may involve growing a hostcell capable of producing immunoglycoproteins in a volume of at leastabout 75 or 100 liters of culture medium comprising castanospermine at aconcentration between about 25 μM and about 800 μM. In some embodiments,methods involve growing a host cell capable of producingimmunoglycoproteins in a volume of at least about 150, 200, 300, 400,500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500,5000, 5500, 6000, 6500, 7000, 7500, 8000, or 10,000 or more liters ofculture medium comprising a carbohydrate modifier, e.g., castanospermineat a concentration between about 25 μM and about 800 μM. In relatedembodiments, any of the preceding compositions may compriseimmunoglycoprotein molecules and any of the foregoing volumes of culturemedia, e.g., essentially serum-free media, and optionally furthercomprise host cells capable of producing the immunoglycoproteinmolecules.

In some embodiments, any of the preceding compositions may comprise atleast about 50, 75, or 100 g of immunoglycoprotein molecules, optionallywith a pharmaceutically acceptable carrier or diluent. In exemplaryembodiments, the composition may comprise at least about 125, 150, 175,200, 300, 400, 500, 600, 700, 800, 900, 1000 or more grams ofimmunoglycoprotein molecules, optionally with a pharmaceuticallyacceptable carrier or diluent. Such compositions may be sterile, or maybe in a container or kit as described herein.

In another aspect, the invention provides a method for increasing theantibody-dependent cytoxicity (ADCC) of immunoglycoprotein moleculesproduced by a host cell, by growing the host cell in culture mediumcomprising castanospermine at a concentration between about 25 and about800 μM, or between about 100 and about 500 μM, or between about 100 andabout 400 μM, or between about 100 and about 300 μM. In exemplaryembodiments, the ADCC is increased at least 2-fold, 3-fold, 4-fold or5-fold.

In related embodiments, the invention provides a method for increasingthe CD16 binding of immunoglycoprotein molecules produced by a hostcell, by growing the host cell in culture medium comprisingcastanospermine at a concentration between about 25 and about 800 μM, orbetween about 100 and about 500 μM, or between about 100 and about 400μM, or between about 100 and about 300 μM. In exemplary embodiments, theCD16 binding is increased by at least 50%, 75%, 100%, 125%, 150%, 175%or 200%.

In the methods of the invention, cell growth, viability and/or densityis not significantly affected (e.g. remains at least 80% or higher ofuntreated cells). The level of immunoglycoprotein production in theculture medium may be at least 100 μg/mL, 125 μg/mL, or 150 μg/mL.

In any of the preceding embodiments the culture medium may beessentially serum-free, and may include a second carbohydrate modifier.

The invention also contemplates compositions comprising any of theimmunoglycoprotein molecules described herein, optionally with a sterilepharmaceutically acceptable carrier or diluent. Such compositions may beadministered in methods of killing or inhibiting growth of cancer cellswhich express on their surface a molecule bound by saidimmunoglycoprotein molecules, or in methods of depleting cells thatexpress on their surface a molecule bound by said immunoglycoproteinmolecules.

In another aspect, methods of the invention may generally involveculturing host cells producing the immunoglycoproteins in culture mediacontaining an appropriate concentration of carbohydrate modifier, e.g.castanospermine, and provide an advantage of improving effector functionwithout significantly affecting cell growth or protein productionlevels. Exemplary immunoglycoproteins that can be manufactured using themethods of the invention include immunoglobulins and small, modularimmunopharmaceutical (SMIP™) products. Such binding molecules preparedaccording to the methods of the invention advantageously retainsubstantially the same properties of binding to target and resultingdirect biological activity, but exhibit improved effector-mediatedfunctions.

In one aspect, the invention provides a method for improving theantibody-dependent cytoxicity (ADCC) and/or the Fc receptor binding ofimmunoglycoproteins produced by a host cell. Such methods involvegrowing the host cell in a volume of at least 750 mL, 1 L, 2 L, 3 L, 4L, 5 L, 10 L, 15 L, 20 L or more of culture medium comprising acarbohydrate modifier, e.g., castanospermine, at a concentration thatincreases the ADCC activity and/or Fc receptor binding of a compositionof immunoglycoprotein molecules produced by the host cell. While theoptimal concentration of such carbohydrate modifier, e.g.,castanospermine, depends on the potency of the carbohydrate modifier andthe relative modulation of ADCC desired, exemplary final concentrationsof carbohydrate modifiers in the culture media are less than 800 μM, orless than 750, 700, 650, 600, 550, 500, 450, 400, 350, 300, 250, 200,150, 125, 100, 90, 80, 70, 60, 50, 40, 30, 20, or 10 μM.

The relative effect on ADCC may be modulated by altering theconcentration or duration of the carbohydrate modifier, e.g.,castanospermine, applied to the cell culture, providing an additionaladvantage compared to conventional methods of improving ADCC by alteringglycosylation. ADCC activity may be measured and expressed using assaysknown in the art and in exemplary embodiments increases by at least 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 2-fold, 3-fold, 4-fold, 5-fold,6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold or 20-fold.

Glycosylation and carbohydrate content is known to affect a variety ofimmunoglobulin effector-mediated functions, including ADCC, CDC andcirculating half-life. The data described herein show that the methodsof the invention and the compositions of the invention are surprisinglyable to provide immunoglycoproteins that exhibit improved ADCC withoutaffecting CDC or half-life. Thus, in exemplary embodiments, ADCC of theimmunoglycoprotein molecule composition is increased but otherimmunoglobulin-type effector functions, such as complement-dependentcytoxicity (CDC) and/or prolonged circulating half-life, remain similaror are not significantly affected (e.g., less than 2-fold increase ordecrease, or less than 50%, 40%, 30% , 20% or 10% increase or decrease).

The Fc receptor binding of the composition of immunoglycoproteinmolecules may be determined as the relative ratio of carbohydratemodifier-treated immunoglycoprotein molecules, vs. untreatedimmunoglycoprotein molecules, that bind to CD16. Exemplary assays aredescribed below in the examples. Fc receptor binding in exemplaryembodiments increases by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, 2-fold, 3-fold, 4-fold, 5-fold or 6-fold. Animmunoglycoprotein composition produced by host cells treated withcarbohydrate modifier, e.g., castanospermine, according to the inventionwill bind to CD16 (high and low affinity forms, i.e. V or F at aminoacid 158) and/or CD32 a or b and/or CD64 with greater affinity in FcRbinding assays than immunoglycoprotein compositions produced by hostcells not so treated. This increase in Fc receptor binding affinity isshown herein to correlate to an increase in ADCC activity.

The invention also provides methods for altering the carbohydratecontent/glycosylation pattern and/or decreasing the fucose content ofimmunoglycoproteins by growing the host cell in a volume of at least 750mL, 1 L, 2 L, 3 L, 4 L, 5 L, 10 L, 15 L, 20 L, 50 L, 100 L, 150 L, 200L, 250 L, 300 L, 350 L, 400 L, 500 L, 600 L, 700 L, 800 L, 900 L, 1000L, 10,000 L or more of culture medium comprising a carbohydratemodifier, e.g., castanospermine, at a concentration that decreases thetotal fucose content and/or alters the glycosylation pattern of acomposition of immunoglycoprotein molecules produced by the host cell.Exemplary final concentrations of carbohydrate modifiers, e.g.,castanospermine, in the culture media are less than 800 μM, or less than750, 700, 650, 600, 550, 500, 450, 400, 350, 300, 250, 200, 150, 125,100, 90, 80, 70, 60, 50, 40, 30, 20, or 10 μM.

The relative effect on fucose content may also be modulated by alteringthe concentration or duration of the carbohydrate modifier, e.g.,castanospermine, applied to the cell culture. The total fucose contentof a composition of the invention may be expressed as the relative ratioor percentage of non-fucosylated immunoglycoprotein molecules to thetotal number of immunoglycoprotein molecules in a composition. Exemplarycompositions contain at least 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95% or more non-fucosylated molecules. The fucose content ofan immunoglycoprotein composition produced by host cells treated withcarbohydrate modifier, e.g., castanospermine, according to the inventionwill be reduced at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold,8-fold, 9-fold or 10-fold or more compared to compositions produced byhost cells not so treated.

In any of the foregoing methods, the host cells may exhibit high levelsof growth during exposure to carbohydrate modifiers, e.g.,castanospermine. For example, an exemplary population doubling time ofCHO cells producing immunoglycoproteins is about 24 hours; aconcentration of carbohydrate modifier according to the invention (e.g.a concentration effective to increase ADCC) is not expected to decreasesuch doubling time. Ideally, an effective concentration of carbohydratemodifier, e.g., castanospermine, does not reduce cell growth by morethan 10, 20, 30, 40, 50, 60 or 70% at a time point 72 hours afteraddition of the carbohydrate modifier.

In any of the foregoing methods, the host cells may exhibit high levelsof protein production during exposure to carbohydrate modifiers, e.g.,castanospermine. For example, protein production levels in the presenceof an effective concentration of carbohydrate modifier, e.g.,castanospermine, may be about 50 μg/mL or higher, or about 75, 100, 125,or 150 μg/mL, or higher. Preferably the host cells exhibit both highlevels of growth and high levels of protein production.

Any culture media known in the art, including essentially serum-freeculture media, may be used. Fed batch, continuous feed, and other typesof culturing methods known in the art may also be used with the methodsof the invention. The carbohydrate modifiers may be added to the seedtrain, to the initial batch culture medium, after a rapid growth phase,or continuously with culture medium (e.g. during continuous feeding).For example, the carbohydrate modifier may be added to an early seedtrain or feedstock at a 10× or 100× concentration, such that subsequentadditions of culture media dilute the concentration of carbohydratemodifier to a level that is still effective in achieving improved ADCCof the recombinant products. Alternatively, the carbohydrate modifier atan effective concentration is included in all culture media added to thecells, obviating the need for dilution. In either case, the carbohydratemodifier is added relatively early in the cell culturing process and aneffective concentration is maintained throughout the culturing processin order to optimize homogeneity of the immunoglycoprotein. The effectof carbohydrate modifiers is believed to be long-lasting, and cancontinue to be observed at least 11-12 days after a one-time addition ofcarbohydrate modifier.

Exemplary carbohydrate modifiers include core glycosylation inhibitors,terminal glycosylation inhibitors, mannosidase inhibitors, and/or earlystage carbohydrate modifiers, and optionally include or excludefucosylation-specific inhibitors, and are described in more detailbelow. The invention contemplates that combinations of two or more, orthree or more carbohydrate modifiers may provide added benefits.Castanospermine is one specifically contemplated carbohydrate modifier.

In another aspect, any of the compositions of the invention provideimmunoglycoprotein molecules that preferably have a binding affinity Kdof at least 10⁷ M⁻¹, or at least 10⁸ M⁻¹, or 10⁹ M⁻¹ for a targetmolecule. Such compositions may comprise one or more sterilepharmaceutically acceptable carriers or diluents.

In a further aspect, the invention provides therapeutic methodsinvolving administration of any of the compositions described herein tosubjects that would benefit from such administration, e.g. sufferingfrom a disorder mediated by cells expressing the target molecule, orsuffering from a type of cancer in which the cancer cells express thetarget molecule on their surface. The invention also contemplates use ofsuch compositions in methods of depleting cells expressing the targetmolecule on their surface. Where the target is CD37, the inventionspecifically contemplates a method of inhibiting cancer cell growth ordestroying cancer cells comprising the step of administering to asubject a composition comprising anti-CD37 SMIP products producedaccording to the methods of the invention. Similarly, where the targetis CD20, the invention specifically contemplates a method of inhibitingcancer cell growth or destroying cancer cells comprising the step ofadministering to a subject a composition comprising anti-CD20 SMIPproducts produced according to the methods of the invention. In relatedembodiments, methods of treating cancer involving arresting or reversingcancer progression are contemplated. The invention further providesmethods of treating autoimmune or inflammatory diseases by administeringanti-CD37 or anti-CD20 SMIP products produced according to the methodsof the invention. In related aspects, the invention contemplates use ofthe glycoprotein compositions of the invention, optionally comprising asterile carrier or diluent, in preparation of a medicament for treatingany of the diseases or disorders described herein.

Immunoglycoproteins

The term “immunoglycoprotein” refers to a glycosylated polypeptide thatbinds to a target molecule and contains sufficient amino acid sequencederived from a constant region of an immunoglobulin to provide aneffector function, preferably ADCC and/or CDC. Exemplary molecules willcontain a sequence derived from a CH2 domain of an immunoglobulin, orCH2 and CH3 domains derived from one or more immunoglobulins. Specificsubsets of immunoglycoproteins contemplated for production according tothe invention include single chain proteins which optionally dimerizethrough covalent or non-covalent associations in the hinge and/or CH3domains. This subset of single chain proteins excludes the typicaltetrameric conformation of immunoglobulins (due to the absence of lightchains) but includes Fc-ligand or Fc-soluble receptor fusions. Specificexamples of single chain proteins include SMIP products.

SMIP products and methods of producing them have been describedpreviously in co-owned U.S. application Ser. No. 10/627,556, and USPatent Publications 2003/133939, 2003/0118592, and 2005/0136049, each ofwhich are incorporated herein by reference in their entirety.Single-Chain Multivalent Binding Proteins with Effector Function aredescribed in International Patent Application No. PCT/US07/71052, filedJun. 12, 2007 (claiming the benefit of U.S. Ser. No. 60/813,261, filedJun. 12, 2006 and 60/853,287, filed Oct. 20, 2006), each of which areincorporated herein by reference in their entirety. SMIP products arenovel binding domain-immunoglobulin fusion proteins that feature abinding domain for a cognate structure such as an antigen, acounterreceptor or the like; an IgG1, IgA or IgE hinge regionpolypeptide or a mutant IgG1 hinge region polypeptide having eitherzero, one or two cysteine residues; and immunoglobulin CH2 and CH3domains. In one embodiment, the binding domain molecule has one or twocysteine residues. In a related embodiment, it is contemplated that whenthe binding domain molecule comprises two cysteine residues, the firstcysteine, which is typically involved in binding between the heavy chainand light chain variable regions, is not deleted or substituted with anamino acid. SMIPs products are capable of ADCC and/or CDC but may becompromised in their ability to form disulfide-linked multimers.Exemplary SMIP products may have one or more binding regions, such as abinding region of an immunoglobulin superfamily member of a variablelight chain and/or variable heavy chain binding region derived from animmunoglobulin. In exemplary embodiments these regions are separated bylinker peptides, which may be any linker peptide known in the art to becompatible with domain or region joinder. Exemplary linkers are linkersbased on the Gly4Ser linker motif, such as (Gly4Ser)n, where n=3-5.

As described in International Patent Application No. PCT/US07/71052,filed Jun. 12, 2007, incorporated herein by reference in its entirety,multivalent single-chain binding proteins with effector function maycomprise a first binding domain derived from an immunoglobulin (e.g., anantibody) or an immunoglobulin-like molecule, a constant sub-regionproviding an effector function, the constant sub-region locatedC-terminal to the first binding domain; a scorpion linker locatedC-terminal to the constant sub-region; and a second binding domainderived from an immunoglobulin (such as an antibody) orimmunoglobulin-like molecule, located C-terminal to the constantsub-region; thereby localizing the constant sub-region between the firstbinding domain and the second binding domain. The single-chain bindingprotein may be multispecific, e.g., bispecific in that it could bind twoor more distinct targets, or it may be monospecific, with two bindingsites for the same target. Moreover, all of the domains of the proteinare found in a single chain, but the protein may form homo-multimers,e.g., by interchain disulfide bond formation. In some embodiments, thefirst binding domain and/or the second binding domain is/are derivedfrom variable regions of light and heavy immunoglobulin chains from thesame, or different, immunoglobulins (e.g., antibodies).

Scorpion linkers comprise at least about 5 amino acids attached to theimmunoglobulin constant sub-region and attached to the second bindingdomain, thereby localizing the scorpion linker between the constantsub-region and the second binding domain. Typically, the scorpion linkerpeptide length is between 5-45 amino acids. Scorpion linkers includehinge-like peptides derived from immunoglobulin hinge regions, such asIgG1, IgG2, IgG3, IgG4, IgA, and IgE hinge regions. Preferably, ahinge-like scorpion linker will retain at least one cysteine capable offorming an interchain disulfide bond under physiological conditions.Scorpion linkers derived from IgG1 may have 1 cysteine or two cysteines,and will preferably retain the cysteine corresponding to an N-terminalhinge cysteine of IgG1. Non-hinge-like peptides are also contemplated asscorpion linkers, provided that such peptides provide sufficient spacingand flexibility to provide a single-chain protein capable of forming twobinding domains, one located towards each protein terminus (N and C)relative to a more centrally located constant sub-region domain.Exemplary non-hinge-like scorpion linkers include peptides from thestalk region of type II C-lectins, such as the stalk regions of CD69,CD72, CD94, NKG2A and NKG2D.

Exemplary SMIP products that can be produced according to the inventioninclude products that bind CD20 or CD37. SMIP products that bind CD20 orCD37 and that comprise specific binding sequences and/or amino acidmodifications are described in co-owned, co-pending U.S. applicationSer. Nos. 10/627,556 and 11/493,132, each hereby incorporated byreference in its entirety.

The CH2 domain of immunoglobulins corresponds to amino acidsAla231-Lys340 of the immunoglobulin heavy chain, using Kabat numbering.As used herein, a “CH2-derived domain” means a domain based on orderived from an immunoglobulin CH2 domain that retains one or moreeffector functions of the domain, e.g. ADCC and/or CDC. Such CH2-deriveddomains will retain the N-linked glycosylation site at Asn297, usingKabat numbering. Exemplary CH2-derived domains retain at least 30%, 40%,50%, 60%, 70%, 80%, or 90% amino acid identity to CH2 of animmunoglobulin, e.g. a human immunoglobulin or human consensusimmunoglobulin sequence. Regions involved in FcR binding for ADCC andCDC have been evaluated in the art. Regions that may be modified withoutsignificantly affecting ADCC are generally disclosed in the art, e.g.,U.S. Pat. No. 6,737,056. Insertions, deletions, or substitutions,including conservative substitutions may be made within the CH2 domainprovided that it retains effector function and retains the Asn297N-linked glycosylation site. Methods for engineering and testing suchmutations are well known in the art.

Other examples of immunoglycoproteins include binding domain-Ig fusions,wherein the binding domain may be a non-naturally occurring peptide or afragment of a naturally occurring ligand or receptor. In the case ofreceptors, fragments of the extracellular domain are preferred.Exemplary fusions with immunoglobulin or Fc regions include: etanerceptwhich is a fusion protein of sTNFRII with the Fc region (U.S. Pat. No.5,605,690), alefacept which is a fusion protein of LFA-3 expressed onantigen presenting cells with the Fc region (U.S. Pat. No. 5,914,111), afusion protein of Cytotoxic T Lymphocyte-associated antigen-4 (CTLA-4)with the Fc region [J. Exp. Med., 181, 1869 (1995)], a fusion protein ofinterleukin 15 with the Fc region [J. Immunol., 160, 5742 (1998)], afusion protein of factor VII with the Fc region [Proc. Natl. Acad. Sci.USA, 98, 12180 (2001)], a fusion protein of interleukin 10 with the Fcregion [J. Immunol., 154, 5590 (1995)], a fusion protein of interleukin2 with the Fc region [J. Immunol., 146, 915 (1991)], a fusion protein ofCD40 with the Fc region [Surgery, 132, 149 (2002)], a fusion protein ofFlt-3 (fms-like tyrosine kinase) with the antibody Fc region [Acta.Haemato., 95, 218 (1996)], a fusion protein of OX40 with the antibody Fcregion [J. Leu. Biol., 72, 522 (2002)], other CD molecules [e.g., CD2,CD30 (TNFRSF8), CD95 (Fas), CD106 (VCAM-1), CD137], adhesion molecules[e.g., ALCAM (activated leukocyte cell adhesion molecule), cadherins,ICAM (intercellular adhesion molecule)-1, ICAM-2, ICAM-3], cytokinereceptors [e.g., interleukin-4R, interleukin-5R, interleukin-6R,interleukin-9R, interleukin-10R, interleukin-12R, interleukin-13Rα],interleukin-13Rα2, interleukin-15R, interleukin-21R], chemokines, celldeath-inducing signal molecules [e.g., B7-H1, DR6 (Death receptor 6),PD-1 (Programmed death-1), TRAIL R1], costimulating molecules [e.g.,B7-1, B7-2, B7-H2, ICOS (inducible co-stimulator)], growth factors[e.g., ErbB2, ErbB3, ErbB4, HGFR], differentiation-inducing factors(e.g., B7-H3), activating factors (e.g., NKG2D), signal transfermolecules (e.g., gp130).

Yet other examples of immunoglycoproteins include antibodies. The term“antibody” herein is defined to include fully assembled antibodies,monoclonal antibodies, polyclonal antibodies, multispecific antibodies(e.g., bispecific antibodies), antibody fragments that can bind antigen(e.g., Fab′, F′(ab)2, Fv, single chain antibodies, diabodies), andrecombinant peptides comprising the forgoing as long as they exhibit thedesired antigen-binding activity. Multimers or aggregates of intactmolecules and/or fragments, including chemically derivatized antibodies,are contemplated. Antibodies of any isotype class or subclass, includingIgG, IgM, IgD, IgA, and IgE, IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2, arecontemplated. Different isotypes have different effector functions; forexample, IgG1 and IgG3 isotypes have antibody-dependent cellularcytotoxicity (ADCC) activity.

An “immunoglobulin” or “native antibody” is a tetrameric glycoproteincomposed of two identical pairs of polypeptide chains (two “light” andtwo “heavy” chains). The amino-terminal portion of each chain includes a“variable” (“V”) region of about 100 to 110 or more amino acidsprimarily responsible for antigen recognition. Within this variableregion, the “hypervariable” region or “complementarity determiningregion” (CDR) consists of residues 24-34 (L1), 50-56 (L2) and 89-97 (L3)in the light chain variable domain and 31-35 (H1), 50-65 (H2) and 95-102(H3) in the heavy chain variable domain as described by Kabat et al.,Sequences of Proteins of Immunological Interest, 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md. (1991)] and/orthose residues from a hypervariable loop (i.e., residues 26-32 (L1),50-52 (L2) and 91-96 (L3) in the light chain variable domain and 26-32(H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variable domain asdescribed by [Chothia et al., J. Mol. Biol. 196: 901-917 (1987)].

The carboxy-terminal portion of each chain contains a constant region.Light chains have a single domain within the constant region. Thus,light chains have one variable region and one constant region domain.Heavy chains have several domains within the constant region. The heavychains in IgG, IgA, and IgD antibodies have three constant regiondomains, which are designated CH1, CH2, and CH3, and the heavy chains inIgM and IgE antibodies have four constant region domains, CH1, CH2, CH3and CH4. Thus, heavy chains have one variable region and three or fourconstant regions.

The heavy chains of immunoglobulins can also be divided into threefunctional regions: the Fd region (a fragment comprising VH and CH1,i.e., the two N-terminal domains of the heavy chain), the hinge region,and the Fc region (the “fragment crystallizable” region, derived fromconstant regions and formed after pepsin digestion). The Fd region incombination with the light chain forms an Fab (the “fragmentantigen-binding”). Because an antigen will react stereochemically withthe antigen-binding region at the amino terminus of each Fab the IgGmolecule is divalent, i.e., it can bind to two antigen molecules. The Fcregion contains the domains that interact with immunoglobulin receptorson cells and with the initial elements of the complement cascade. Thus,the Fc fragment is generally considered responsible for the effectorfunctions of an immunoglobulin, such as complement fixation and bindingto Fc receptors.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations or alternativepost-translational modifications that may be present in minor amounts,whether produced from hybridomas or recombinant DNA techniques.Nonlimiting examples of monoclonal antibodies include murine, chimeric,humanized, or human antibodies, or variants or derivatives thereof.

Humanizing or modifying antibody sequence to be more human-like isdescribed in, e.g., Jones et al., Nature 321:522 525 (1986); Morrison etal., Proc. Natl. Acad. Sci., U.S.A., 81:6851 6855 (1984); Morrison andOi, Adv. Immunol., 44:65 92 (1988); Verhoeyer et al., Science 239:15341536 (1988); Padlan, Molec. Immun. 28:489 498 (1991); Padlan, Molec.Immunol. 31(3):169 217 (1994); and Kettleborough, C. A. et al., ProteinEng. 4(7):773 83 (1991); Co, M. S., et al. (1994), J. Immunol. 152,2968-2976); Studnicka et al. Protein Engineering 7: 805-814 (1994); eachof which is incorporated herein by reference.

One method for isolating human monoclonal antibodies is the use of phagedisplay technology. Phage display is described in e.g., Dower et al., WO91/17271, McCafferty et al., WO 92/01047, and Caton and Koprowski, Proc.Natl. Acad. Sci. USA, 87:6450-6454 (1990), each of which is incorporatedherein by reference. Another method for isolating human monoclonalantibodies uses transgenic animals that have no endogenousimmunoglobulin production and are engineered to contain humanimmunoglobulin loci. See, e.g., Jakobovits et al., Proc. Natl. Acad.Sci. USA, 90:2551 (1993); Jakobovits et al., Nature, 362:255-258 (1993);Bruggermann et al., Year in Immuno., 7:33 (1993); WO 91/10741, WO96/34096, WO 98/24893, or U.S. patent application publication nos.20030194404, 20030031667 or 20020199213; each incorporated herein byreference.

Antibody fragments may be produced by recombinant DNA techniques or byenzymatic or chemical cleavage of intact antibodies. “Antibodyfragments” comprise a portion of an intact full length antibody,preferably the antigen binding or variable region of the intactantibody, and include multispecific (bispecific, trispecific, etc.)antibodies formed from antibody fragments. Nonlimiting examples ofantibody fragments include Fab, Fab′, F(ab′)2, Fv [variable region],domain antibody (dAb) [Ward et al., Nature 341:544-546, 1989],complementarity determining region (CDR) fragments, single-chainantibodies (scFv) [Bird et al., Science 242:423-426, 1988, and Huston etal., Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988, optionally includinga polypeptide linker; and optionally multispecific, Gruber et al., J.Immunol. 152: 5368 (1994)], single chain antibody fragments, diabodies[EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl. Acad. Sci.USA, 90:6444-6448 (1993)], triabodies, tetrabodies, minibodies [Olafsen,et al., Protein Eng Des Sel. 2004 April; 17(4):315-23], linearantibodies [Zapata et al., Protein Eng., 8(10):1057-1062 (1995)];chelating recombinant antibodies [Neri et al., J Mol Biol. 246:367-73,1995], tribodies or bibodies [Schoonjans et al., J Immunol. 165:7050-57,2000; Willems et al., J Chromatogr B Analyt Technol Biomed Life Sci.786:161-76, 2003], intrabodies [Biocca, et al., EMBO J. 9:101-108, 1990;Colby et al., Proc Natl Acad Sci USA. 101:17616-21, 2004], nanobodies[Cortez-Retamozo et al., Cancer Research 64:2853-57, 2004], anantigen-binding-domain immunoglobulin fusion protein, a camelizedantibody [Desmyter et al., J. Biol. Chem. 276:26285-90, 2001; Ewert etal., Biochemistry 41:3628-36, 2002; U.S. Patent Publication Nos.20050136049 and 20050037421], a VHH containing antibody, mimetibodies[U.S. Patent Publication Nos. 20050095700 and 20060127404; WO 04/002424A2; WO 05/081687 A2], or variants or derivatives thereof, andpolypeptides that contain at least a portion of an immunoglobulin thatis sufficient to confer specific antigen binding to the polypeptide,such as a CDR sequence, as long as the antibody retains the desiredantigen-binding activity.

The term “variant” when used in connection with antibodies refers topolypeptide sequence of an antibody that contains at least one aminoacid substitution, deletion, or insertion in the variable region or theportion equivalent to the variable region, provided that the variantretains the desired target binding affinity or biological activity. Inaddition, the antibodies of the invention may have amino acidmodifications in the constant region to modify effector function of theantibody, including half-life or clearance, ADCC and/or CDC activity.Such modifications can enhance pharmacokinetics or enhance theeffectiveness of the antibody in treating cancer, for example. In thecase of IgG1, modifications to the constant region, particularly thehinge or CH2 region, may increase or decrease effector function,including ADCC and/or CDC activity. In other embodiments, an IgG2constant region is modified to decrease antibody-antigen aggregateformation. In the case of IgG4, modifications to the constant region,particularly the hinge region, may reduce the formation ofhalf-antibodies.

The term “derivative” when used in connection with antibodies refers toantibodies covalently modified by conjugation to therapeutic ordiagnostic agents, labeling (e.g., with radionuclides or variousenzymes), covalent polymer attachment such as pegylation (derivatizationwith polyethylene glycol) and insertion or substitution by chemicalsynthesis of non-natural amino acids. Derivatives of the invention willretain the binding properties of underivatized molecules of theinvention. Conjugation of cancer-targeting antibodies to cytotoxicagent, for example, radioactive isotopes (e.g., I131, I125, Y90 andRe186), chemotherapeutic agents, or toxins, may enhance destruction ofcancerous cells.

An immunoglycoprotein that is “specific” for a target molecule binds tothat target with a greater affinity than any other target.Immunoglycoproteins of the invention may have affinities for theirtargets of a Ka of at least about 10⁴ M⁻¹, or alternatively of at leastabout 10⁵ M⁻¹, 10⁶ M⁻¹, 10⁷ M⁻¹, 10⁸ M⁻¹, 10⁹ M⁻¹, or 10¹⁰ M⁻¹. Suchaffinities may be readily determined using conventional techniques, suchas by using a BIAcore instrument or by radioimmunoassay usingradiolabeled target antigen. Affinity data may be analyzed, for example,by the method of Scatchard et al., Ann N.Y. Acad. Sci., 51:660 (1949).

Carbohydrate Modifiers

A “carbohydrate modifier” is a small organic compound, preferably ofmolecular weight<1000 daltons, that inhibits the activity of an enzymeinvolved in the addition, removal, or modification of sugars that arepart of a carbohydrate attached to a polypeptide. Glycosylation is anextremely complex process that takes place in the endoplasmic reticulum(“core glycosylation”) and in the Golgi bodies (“terminalglycosylation”).

Other polypeptide-based or polynucleotide-based repressors ofglycosylation enzymes, including RNAi or antisense that inhibitsactivity of early stage carbohydrate modifiers, are useful according tothe invention but are excluded from the definition of “carbohydratemodifier.”

As used herein, “early stage carbohydrate modifier” refers to aninhibitor of one or more of the glycosylation steps prior to addition ofN-acetylglucosamine to mannose, including ER glucosidase I, ERglucosidase II, ER mannosidase, Golgi mannosidase IA, Golgi mannosidaseIB, Golgi mannosidase IC and GlcNAc-transferase I.

Subsequent glycosylation steps include Golgi mannosidase II,GlcNAc-transferase II, galactosyl transferase and sialyl transferase,fucosyl transferase, and fucokinase.

Exemplary carbohydrate modifiers include any of the following.Castanospermine is believed to be a glucosidase I and II inhibitor.Deoxyfuconojirimycin is a fucosidase inhibitor.6-Methyl-tetrahydro-pyran-2H-2,3,4-triol has been reported in vitro toinhibit phosphorylation of L-fucose, the first step in biosynthesis ofGDP-L-Fucose. 6,8a-diepi-castanospermine is a reportedfucosyltransferase inhibitor. 1-N-iminosugars A and B (also known as1-Butyl-5-methyl-piperidine-3,4-diol hydrochloride and5-Methyl-piperidine-3,4-diol hydrochloride, respectively) have beenreported to be fucosyltransferase inhibitors. Deoxymannojirimycin (DMJ)is an ER mannosidase I inhibitor. Kifunensine (Kf) is an ER mannosidaseI inhibitor. Swainsonine (Sw) is a golgi mannosidase II inhibitor.Monensin (Mn) is an inhibitor of intracellular protein transport betweenER and Golgi that interferes with elongation of core oligosaccharide.

Data described herein show that a variety of glycosidase and/ormannosidase inhibitors provide one or more of desired effects ofincreasing ADCC activity, increasing Fc receptor binding, and alteringglycosylation pattern.

In exemplary embodiments, castanospermine (MW 189.21) is added to theculture medium to a final concentration of about 200 μM (correspondingto about 37.8 μg/mL), or concentration ranges greater than about 10, 20,30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 μM, and upto about 300, 275, 250, 225, 200, 175, 150, 125, 100, 75, 60, or 50μg/mL. For example, ranges of 10-50, or 50-200, or 50-300, or 100-300,or 150-250 μM are contemplated.

In other exemplary embodiments, DMJ, for example DMJ-HCl (MW 199.6) isadded to the culture medium to a final concentration of about 200 μM(corresponding to about 32.6 μg DMJ/mL), or concentration ranges greaterthan about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140,or 150 μM, and up to about 300, 275, 250, 225, 200, 175, 150, 125, 100,75, 60, or 50 μg/mL. For example, ranges of 10-50, or 50-200, or 50-300,or 100-300, or 150-250 μM are contemplated.

In other exemplary embodiments, kifunensine (MW 232.2) is added to theculture medium to a final concentration of about 10 μM (corresponding toabout 2.3 μg/mL), or concentration ranges greater than about 0.5, 1, 2,3, 4, 5, 6, 7, 8, 9 or 10 μM, and up to about 50, 45, 40, 35, 30, 25,20, 19, 18, 17, 16, 15, 14, 13, 12, or 11 μM. For example, ranges of1-10, or 1-25, or 1-50, or 5-10, or 5-25, or 5-15 μM are contemplated.

Recombinant Constructs, Cells and Culturing Methods

As used herein, “host cell” specifically excludes rodent hybridomas butincludes any other cell that is capable of glycosylation (i.e. additionof carbohydrate to an amino acid of a polypeptide) and that has beenmodified through recombinant means to express increased levels of aprotein product. Progeny of host cells that retain the recombinantmodification and the ability to express the protein product are includedwithin the term “host cell”.

Exemplary elements of expression vectors or regulatory sequences mayinclude an origin of replication, a promoter, an operator, or otherelements that mediate transcription and translation. Promoters can beconstitutive or active and may further be cell type specific, tissuespecific, individual cell specific, event specific, temporally specificor inducible. Event specific promoters are active or up regulated onlyupon the occurrence of an event. In addition to the promoter, repressorsequences, negative regulators, or tissue-specific silencers may beinserted to reduce non-specific expression. Other elements includeinternal ribosome binding sites, a transcription terminator sequence,including a polyadenylation sequence, splice donor and acceptor sites,and an enhancer, a selectable marker and the like.

The culture medium can include any necessary or desirable ingredientsknown in the art, such as carbohydrates, including glucose, essentialand/or non-essential amino acids, lipids and lipid precursors, nucleicacid precursors, vitamins, inorganic salts, trace elements includingrare metals, and/or cell growth factors. The culture medium may bechemically defined or may include serum, plant hydrolysates, or otherderived substances. The culture medium may be essentially or entirelyserum-free or animal-component free. “Essentially serum-free” means thatthe medium lacks any serum or contains an insignificant amount of serum.Exemplary supplementary amino acids depleted during cell culture includeasparagine, aspartic acid, cysteine, cystine, isoleucine, leucine,tryptophan, and valine.

Commercially available lipids and/or lipid precursors include choline,ethanolamine, or phosphoethanolamine, cholesterol, fatty acids such asoleic acid, linoleic acid, linolenic acid, methyl esters,D-alpha-tocopherol, e.g. in acetate form, stearic acid; myristic acid,palmitic acid, palmitoleic acid; or arachidonic acid. Essential aminoacids include Arginine, Histidine, Isoleucine, Leucine, Lysine,Methionine, Phenylalanine, Threonine, Tryptophan and Valine.Non-essential amino acids include Alanine, Asparagine, Aspartate,Cysteine, Glutamate, Glutamine, Glycine, Proline, Serine, and Tyrosine.Commercially available inorganic or trace elements, supplied asappropriate salts, include sodium, calcium, potassium, magnesium,copper, iron, zinc, selenium, molybdenum, vanadium, manganese, nickel,silicon, tin, aluminum, barium, cadmium, chromium, cobalt, germanium,potassium, silver, rubidium, zirconium, fluoride, bromide, iodide andchloride. The medium may also optionally include a nonionic surfactantor surface-active agent to protect the cells from the mixing oraeration. The culture medium may also comprise buffers such as sodiumbicarbonate, monobasic and dibasic phosphates, HEPES and/or Tris. Theculture medium may also comprise inducers of protein production, such assodium butyrate, or caffeine.

The invention also provides methods for producing an immunoglycoproteincomprising culturing a host cell in any of the culture media or underany of the conditions described herein. Such methods may further includethe step of recovering the immunoglycoprotein from the host cells orculture medium. The carbohydrate modifier may be included in the initialculture medium, or may be added during the initial growth phase or atlater phases. When the recombinant protein is secreted into the medium,the medium can be harvested periodically and replaced with fresh mediumthrough several harvest cycles.

Although CHO cells, which are widely used for therapeutic proteinproduction, are preferred, any host cells known in the art to produceglycosylated proteins may be used, including yeast cells, plant cells,plants, insect cells, and mammalian cells. Exemplary yeast cells includePichia, e.g. P. pastoris, and Saccharomyces e.g. S. cerevisiae, as wellas Schizosaccharomyces pombe, Kluyveromyces, K. Zactis, K. fragilis, K.bulgaricus, K. wickeramii, K. waltii, K. drosophilarum, K.thernotolerans, and K. marxianus; K. yarrowia; Trichoderma reesia,Neurospora crassa, Schwanniomyces, Schwanniomyces occidentalis,Neurospora, Penicillium, Totypocladium, Aspergillus, A. nidulans, A.niger, Hansenula, Candida, Kloeckera, Torulopsis, and Rhodotorula.Exemplary insect cells include Autographa californica and Spodopterafrugiperda, and Drosophila. Exemplary mammalian cells include varietiesof CHO, BHK, HEK-293, NS0, YB2/3, SP2/0, and human cells such as PER-C6or HT1080, as well as VERO, HeLa, COS, MDCK, NIH3T3, Jurkat, Saos,PC-12, HCT 116, L929, Ltk-, WI38, CV1, TM4, W138, Hep G2, MMT, aleukemic cell line, embryonic stem cell or fertilized egg cell.

The cells may be cultured in any culture system and according to anymethod known in the art, including T-flasks, spinner and shaker flasks,roller bottles and stirred-tank bioreactors. Anchorage-dependent cellscan also be cultivated on microcarrier, e.g. polymeric spheres, that aremaintained in suspension in stirred-tank bioreactors. Alternatively,cells can be grown in single-cell suspension. Culture medium may beadded in a batch process, e.g. where culture medium is added once to thecells in a single batch, or in a fed batch process in which smallbatches of culture medium are periodically added. Medium can beharvested at the end of culture or several times during culture.Continuously perfused production processes are also known in the art,and involve continuous feeding of fresh medium into the culture, whilethe same volume is continuously withdrawn from the reactor. Perfusedcultures generally achieve higher cell densities than batch cultures andcan be maintained for weeks or months with repeated harvests.

Determination of Oligosaccharide Characteristics

Oligosaccharide content and characteristics of the immunoglycoproteincompositions of the invention can be determined using means known in theart. For example, oligosaccharides can be released from theimmunoglycoprotein by enzymatic digestion with PNGase-F under denaturingconditions. The oligosaccharides can then be derivatized with afluorescent modifier and resolved by normal phase chromatography coupledwith fluorescence detection. Major species can be analyzed by MALDI-TOF.

The presence of N-linked glycosylation sites that are available forlinkage of oligosaccharides can be determined by methods known in theart. For example, mass differences between glycosylatedimmunoglycoprotein and unglycosylated recombinant immunoglycoprotein(e.g., expressed recombinantly in a bacterial cell that does notglycosylate protein) provides information on the total mass ofsaccharide added via glycosylation. Individual mutation of theoreticalN-linked glycosylation sites (the tripeptide consensus motifAsn-X-Thr/Ser) to destroy the motif, followed by comparison of the massof the nonmutated immunoglycoprotein to the mutated immunoglycoprotein,can provide information on whether the site is available forglycosylation. If the hypothetical site is a potential N-linkedglycosylation site, destruction of the motif should yield a reduction inthe carbohydrate content of the immunoglycoprotein.

Monosaccharide composition analysis can be carried out generally asfollows. Monosaccharides are released by acid hydrolysis, thenderivitized with a fluorescent modifier and resolved by reverse phasechromatography coupled with fluorescence detection, and quantified usinglabeled monosaccharide standards. Results can be expressed as the moleratio of each monosaccharide to protein. From this information, theratio of saccharide to N-glycosylation site can be calculated.

Immunoglycoproteins contain sufficient amino acid sequence derived froma constant region of an immunoglobulin to provide an effector function,preferably ADCC and/or CDC. This amino acid sequence derived from theconstant region comprises an N-linked glycosylation site at the positioncorresponding to Asn297, using Kabat numbering. Thus, exemplarymolecules will contain a sequence derived from a CH2 domain of animmunoglobulin that includes the Asn297.

Oligosaccharide characteristics of a particular N-linked glycosylationsite can be determined by producing a fragment containing that N-linkedglycosylation site via enzymatic digestion, and analyzing theoligosaccharide content of the fragment. For example, the ratio ofoligosaccharide or monosaccharide from a CH2-derived domain to N-linkedglycosylation site(s) in this same CH2-derived domain can be determinedas follows. Proteolytic fragments of the immunoglycoprotein can beproduced by enzymatic digestion. Subsequent LCMS analysis of theresolved peptides (MS analysis of peptide map), with comparison to thepeptide map of a deglycosylated molecule, will provide identification ofthe specific peptides which contain the glycosylation site. The glycancontent at each N-linked site can then be deduced by glycoprofiling, inwhich the known peptide mass is subtracted from the determinedglycopeptide mass and the result analyzed by comparison to the mass ofknown glycan structures. In addition, specific regions (e.g., in thecase of multiple glycosylation sites) of the intact molecule can beseparated by enzymatic digest then resolved and collected by HPLC. Theseindividual sites can then be analyzed for glycan content by MS,monosaccharide analysis and oligosaccharide analysis.

In addition, specific monosaccharide branching or content can bedetermined as follows. Mono or oligosaccharides can be released usingspecific enzymes that only recognize certain sugars or branchingpatterns. The sugars released can be labeled and resolved.

LCMS Glycoprofiling can be carried out as generally follows.Glycoprofiling from the single N-linked glycosylation site can beperformed by comparison of native vs. de-glycosylated sample in bothwhole mass and glycopeptide LCMS analysis. Samples are reduced to themonomeric form and analyzed by ESI-TOF. GlycoMod can be used to identifythe glycan species.

Use of Immunoglycoproteins

The immunoglycoproteins of the invention are useful as therapeutics totreat diseases mediated by the target molecule, or, for example, ascytolytic agents to kill cancer cells that have the target moleculeexpressed or associated with the cell surface.

“Treatment” or “treating” refers to either a therapeutic treatment orprophylactic or preventative treatments. A therapeutic treatment mayimprove at least one symptom of disease in an individual receivingtreatment or may delay worsening of a progressive disease in anindividual, or prevent onset of additional associated diseases. Animproved response is assessed by evaluation of clinical criteriawell-known in the art for the disease state.

A “therapeutically effective dose” or “effective dose” of animmunoglycoprotein refers to that amount of the compound sufficient toresult in amelioration of one or more symptoms of the disease beingtreated. When applied to an individual active ingredient, administeredalone, a therapeutically effective dose refers to that ingredient alone.When applied to a combination, a therapeutically effective dose refersto combined amounts of the active ingredients that result in thetherapeutic effect, whether administered in combination, serially orsimultaneously. The doses may be administered based on weight of thepatient, e.g., at a dose of 0.01 to 50 mg/kg, and may be administered ona daily or weekly basis, or every 2 weeks, every 3 weeks, or once amonth.

To administer the immunoglycoproteins of the invention to humans or testanimals, it is preferable to formulate the molecule in a compositioncomprising one or more pharmaceutically acceptable carriers or diluents,preferably sterile carriers or diluents if the composition is forparenteral administration. The phrase “pharmaceutically orpharmacologically acceptable” refer to molecular entities andcompositions that do not produce allergic, or other adverse reactionswhen administered using routes well-known in the art, as describedbelow. “Pharmaceutically acceptable carriers” include any and allclinically useful solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents and thelike. Generally, compositions are also essentially free of pyrogens, aswell as other impurities that could be harmful to the recipient.

Immunoglycoproteins may be administered orally, topically,transdermally, parenterally, by inhalation spray, vaginally, rectally,or by intracranial injection. The term parenteral as used hereinincludes subcutaneous injections, intravenous, intramuscular,intracisternal injection, or infusion techniques. Administration byintravenous, intradermal, intramuscular, intramammary, intraperitoneal,intrathecal, retrobulbar, intrapulmonary injection and or surgicalimplantation at a particular site is contemplated as well.

In one embodiment, administration is performed at the site of a canceror affected tissue needing treatment by direct injection into the siteor via a sustained delivery or sustained release mechanism, which candeliver the formulation internally. For example, biodegradablemicrospheres or capsules or other biodegradable polymer configurationscapable of sustained delivery of a composition (e.g., a solublepolypeptide, antibody, or small molecule) can be included in theformulations of the invention implanted near the cancer

Therapeutic compositions may also be delivered to the patient atmultiple sites. The multiple administrations may be renderedsimultaneously or may be administered over a continuous period of time.

Injection of aqueous solutions are preferred. Aqueous compositions canbe lyophilized for storage and reconstituted in a suitable carrier priorto use. This technique has been shown to be effective with conventionalimmunoglobulins. Any suitable lyophilization and reconstitutiontechniques can be employed. It will be appreciated by those skilled inthe art that lyophilization and reconstitution can lead to varyingdegrees of activity loss and that use levels may have to be adjusted tocompensate.

In all cases the form must be sterile and must be fluid to the extentthat easy syringability exists. The proper fluidity can be maintained,for example, by the use of a coating, such as lecithin, by themaintenance of the required particle size in the case of dispersion andby the use of surfactants. It must be stable under the conditions ofmanufacture and storage and may be preserved against the contaminatingaction of microorganisms, such as bacteria and fungi. The prevention ofthe action of microorganisms can be brought about by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In manycases, it will be desirable to include isotonic agents, for example,sugars or sodium chloride.

In addition, the properties of hydrophilicity and hydrophobicity of thecompositions contemplated for use in the invention are well balanced,thereby enhancing their utility for both in vitro and especially in vivouses, while other compositions lacking such balance are of substantiallyless utility. Specifically, compositions contemplated for use in theinvention have an appropriate degree of solubility in aqueous mediawhich permits absorption and bioavailability in the body, while alsohaving a degree of solubility in lipids which permits the compounds totraverse the cell membrane to a putative site of action.

Also contemplated in the present invention is the administration of animmunoglycoprotein composition in conjunction with a second agent.

As an additional aspect, the invention includes kits or articles ofmanufacture which comprise one or more compounds or compositionspackaged in a manner which facilitates their use to practice methods ofthe invention. In one embodiment, such a kit includes aimmunoglycoprotein described herein, optionally with a secondtherapeutic agent, packaged in a container such as a sealed bottle orvessel, with a label affixed to the container or included in the packagethat describes use of the compound or composition in practicing themethod. Preferably, the compound or composition is packaged in a unitdosage form. The kit may further include a device suitable foradministering the composition according to a specific route ofadministration or for practicing a screening assay. Preferably, the kitcontains a label that describes use of the composition.

The invention further contemplates the use of the immunoglycoproteins ofthe invention in the manufacture of a medicament for the inhibition orprevention or treatment of a disease, condition, or disorder in asubject characterized or mediated by the target to which theimmunoglycoprotein binds.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts cell growth of CHO cells expressing TRU-016 grown in cellmedia with various concentrations of castanospermine, as shown by cellcounts of cells/ml.

FIG. 2 depicts cell viability of CHO cells expressing TRU-016 grown incell media with various concentrations of castanospermine, as shown by %of live cells.

FIG. 3 depicts CD16 binding of TRU-015 produced by cells cultured in thepresence of varying concentrations of castanospermine and showsgeometric mean fluorescent intensity vs. castanospermine concentration.

FIG. 4 depicts CD16 binding, as shown by geometric mean fluorescentintensity, of TRU-016 produced by cells cultured in the presence ofvarious concentrations of 6,8a-diepicastanospermine, swainsonine, ordeoxymannojirimycin (DMJ).

FIG. 5 depicts CD16 binding, as shown by mean fluorescent intensity, ofTRU-016 produced by cells cultured in the presence of varyingconcentrations of kifunensine.

FIG. 6 depicts CD16 binding, as shown by mean fluorescent intensity, ofProtein A-purified TRU-016 produced by cells cultured in the presence ofvarying concentrations of castanospermine.

FIGS. 7 and 8 depict ADCC of TRU-015 measured using PBMC of highaffinity and low affinity donors, respectively, and plots concentrationof TRU-015 added vs. % specific killing.

FIG. 9 depicts ADCC of TRU-016 produced by cells cultured in thepresence of varying concentrations of castanospermine, and plots %specific killing vs. concentration of TRU-016 added.

FIG. 10 depicts ADCC of TRU-016 produced by cells cultured in thepresence of various carbohydrate modifiers, and plots % specific killingvs. concentration of TRU-016 added.

FIG. 11 depicts pharmacokinetic data in mice administered TRU-016produced by cells cultured in the presence of various carbohydratemodifiers.

FIG. 12 depicts CD16 binding of TRU-016 in sera of mice administered theTRU-016 produced by cells treated with various carbohydrate modifiers.

FIG. 13 depicts relative tumor volume at 8 days in mice implanted withtumor cells and administered TRU-016 produced from cells treated withvarious carbohydrate modifiers, or untreated cells.

FIG. 14 depicts % survival of mice implanted with tumor cells andadministered TRU-016 produced from cells treated with variouscarbohydrate modifiers, or untreated cells.

FIG. 15 depicts CDC of TRU-015 produced by cells cultured in thepresence of castanospermine, and plots % propidium iodide positive (deadcells) vs. concentration of TRU-015 test protein.

FIG. 16 depicts CDC of TRU-016 produced by cells cultured in thepresence of various carbohydrate modifiers, and plots % propidium iodidepositive (dead cells) vs. concentration of TRU-016 test protein.

FIG. 17 depicts relative specific protein production of TRU-016 over arange of castanospermine concentrations.

FIG. 18 depicts the results of an assay for simultaneous binding ofTRU-016 to CD37 and FcγRIIIa (CD16) over a range of castanospermineconcentrations.

FIG. 19 depicts dose response binding curves of TRU-016 toCD37-expressing cells for a range of castanospermine concentrations.

FIG. 20 depicts ADCC activity curves of TRU-016 over a range ofcastanospermine concentrations.

FIG. 21 depicts the results of an assay for simultaneous binding ofCHO-K1 produced TRU-016 and CS-generated glycovariants thereof to CD37and FcγRIIIa (CD16). Concentrations of CS utilized for generation of thetested glycovariants are as indicated. Results are depicted as both theraw curve data (A) as well as in a bar graph form that indicates themaximal geometric mean fluorescence intensity achieved by eachglycovariant and the TRU-016 control (B).

FIG. 22 depicts binding of CHO-K1 produced TRU-016 and CS-generatedglycovariants thereof to the CD37 target antigen expressed on Daudicells. Concentrations of CS utilized for generation of the testedglycovariants are as indicated.

FIG. 23 depicts the results of Antibody Dependent Cellular Cytotoxicity(ADCC) assays performed with CHO-K1 produced TRU-016 and CS-generatedglycovariants thereof. Concentrations of CS utilized for generation ofthe tested glycovariants are as indicated. A-C show the different donorPBL effector cell CD16 genotypes used for each experiment. A, homozygoushigh (V/V) binder; B, heterozygous (F/V) binder; C, homozygous low (F/F)binder.

FIG. 24 depicts an assay for simultaneous binding of DG44 CHO producedTRU-016 and CS-generated glycovariants thereof to CD37 and FcγRIIIa(CD16). These samples represented PA samples purified from bioreactorruns. Concentrations of CS utilized for generation of the testedglycovariants are as indicated. Results are depicted as both the rawcurve data (A) as well as in a bar graph form that indicates the maximalgeometric mean fluorescence intensity achieved by each glycovariant andthe TRU-016 control (B).

FIG. 25 depicts an Antibody Dependent Cellular Cytotoxicity (ADCC) assayperformed with DG44 CHO produced TRU-016 and CS-generated glycovariantsthereof. Concentrations of CS utilized for generation of the testedglycovariants are as indicated. These samples represented PA and CHTsamples purified from bioreactor runs. Results are shown as raw datacurves (A) as well as relative fold-improvement compared to the 0 μM CS(control) TRU-016 CHT sample (B).

FIG. 26 depicts results obtained from LCMS tryptic digest glycoprofilingof TRU-016 and CS-generated glycovariants thereof. The top frame showsthat section of the chromatograms in which the glycopeptides elute andincludes all detected peaks. The bottom frame is the same data, butshows only those peaks which correspond to the mass of the glycopeptide.

FIG. 27 depicts results from a monosaccharide analysis of TRU-016 andCS-generated glycovariants thereof. The glycovariant treated with 400 μMCS, the control, and the monosaccharide standards are each indicated byarrows.

FIG. 28 depicts results from an oligosaccharide analysis of TRU-016Control and CS-generated glycovariant products. The relative percent(peak area) of the major species is identified above the correspondingpeaks.

DETAILED DESCRIPTION OF THE INVENTION EXAMPLES Example 1

Production of SMIP Products

TRU-016

CD37-specific SMIPs are described in co-owned U.S. application Ser. No.10/627,556 and U.S. Patent Publication Nos. 2003/133939, 2003/0118592and 2005/0136049, each incorporated by reference herein in its entirety.An exemplary SMIP, TRU-016, is produced as described below. As usedherein, TRU-016 refers to any CD37-specific SMIP.

TRU-016 [G28-1 scFv VH11S (SSC-P) H WCH2 WCH3] is a recombinant singlechain protein that binds to the CD37 antigen. The nucleotide and aminoacid sequences of TRU-016 are respectively set out in SEQ ID NOS: 1 and2. Additional sequences are set forth in co-owned, concurrently filedU.S. patent application Ser. No. 11/493,132 [Entitled “B-CELL REDUCTIONUSING CD37-SPECIFIC AND CD2O-SPECIFIC BINDING MOLECULES”], herebyincorporated by reference in its entirety. The binding domain was basedon the G28-1 antibody sequence previously disclosed in the patentpublications listed in the preceding paragraph, which disclosure isincorporated herein by reference. The binding domain is connected to theeffector domain, the CH2 and CH3 domains of human IgG1, through amodified hinge region. This TRU-016 exists as a dimer in solution.

TRU-016 is produced by recombinant DNA technology in a Chinese hamsterovary (CHO) mammalian cell expression system. TRU-016 SMIPs are purifiedfrom CHO culture supernatants by Protein A affinity chromatography.Using dPBS, a 50 mL rProtein A FF sepharose column (GE HealthcarerProtein A Sepharose FF, Catalog #17-0974-04) is equilibrated at 5.0mls/min (150 cm/hr) for 1.5 column volumes (CV). The culture supernatantis loaded to the rProtein A Sepharose FF column at a flow rate of 1.7mls/min using the AKTA Explorer 100 Air (GE healthcare AKTA Explorer 100Air, Catalog #18-1403-00), capturing the recombinant TRU-016. The columnis washed with dPBS for 5 Column Volumes (CV), then 1.0 M NaCl, 20 mMSodium Phosphate, pH 6.0, and then with 25 mM NaCl, 25 mM NaOAc, pH 5.0.These washing steps remove nonspecifically bound CHO host cell proteinsfrom the rProtein A column that contribute to product precipitationafter elution.

The recombinant TRU-016 is eluted from the column with 100 mM Glycine,pH 3.5. 10 mL fractions of the eluted product were recovered and theeluted product was then brought to pH 5.0 with 20% of the eluted volumeof 0.5 M 2-(N-Morpholino)ethanesulfonic acid (MES) pH 6.0. This elutedproduct is concentrated to approximately 25 mg/mL TRU-016 and filtersterilized.

Purified protein is then subjected to GPC size exclusion chromatography(SEC) to achieve further purification of the TRU-016 (dimer) moleculefrom higher molecular weight aggregates. Using dPBS, an XK 50/100 column(GE healthcare XK 50/100 empty chromatography column, Catalog#18-8753-01) containing 1 L of Superdex 200 FF sepharose is equilibratedat 12.6 mls/min (38 cm/hr) for 1.5 column volumes (CV). A maximum volumeof 54 mls (3% CV) of sample is applied to the column. The columncontinues to run at 12.6 ml/min and the eluted protein is fractionatedin 40 mL fractions. Each fraction is analyzed for product quality usingan analytic HPLC, and the eluted fractions are pooled for >95% POI(non-aggregated) TRU-016. This resultant pool is filter sterilized at0.22 μm. The material is then concentrated and formulated with 20 mMsodium phosphate and 240 mM sucrose, at pH 6.0.

An alternative method for purification of the glycovariant is asfollows. TRU-016 SMIPS are purified from CHO culture supernatants byProtein A affinity chromatography. Using dPBS, a 1 mL MabSelect™affinity chromatography column (GE Healthcare Hitrap MabSelect™, catalog#28-4082-53) is equilibrated at 1.0 mL/min for 7 column volumes (CV).The culture supernatant is loaded on to the MabSelect™ column at aflowrate of 1.0 mL/min using the Akta Explorer 100 Air (GE Healthcare,Akta Explorer 100 Air, catalog #18-1403-00) capturing the recombinantTRU-016. The column is washed with dPBS for 20 CV, then with 20 mMsodium phosphate, 1.0 M NaCl, pH 7.0 for 5 CV and then with dPBS for 3CV.

The recombinant TRU-016 is eluted from the column with 10 mM Citrate, pH3.5 and the column is stripped with 10 mM Citrate 3.0 for 8 CV.Following the strip the column is re-equilibrated for 5 CV with dPBS.The protein is collected into fractions during elution which are pooledbased upon absorbance and this pooled material is brought to pH 5.0 withan addition of approximately 400 μL of 0.55 M2-(N-Morpholin)ethanesulfonic acid (MES) pH 6.0 per 5 mL of elution.This neutralized eluate is filter sterilized and stored at 2-8° C. Thesamples from this purification method are referred to as PA samples.

Experiments may be performed to confirm that the binding specificity ofthe parent antibody to the CD37 cell surface receptor is preserved inTRU-016. Human PBMCs are isolated over LSM density gradients andincubated with unconjugated TRU-016 and PE-conjugated anti-human CD19.Cells are washed and incubated with 1:100 FITC GAH IgG (Fc specific) for45 minutes on ice. Cells are washed and analyzed by two-color flowcytometry on a FACsCalibur instrument using Cell Quest software. Cellsare gated for B lymphocytes or non-B lymphocytes by CD19 staining

With increasing concentrations of TRU-016, the FITC signal on the Blymphocyte (CD19 positive gate) increases rapidly from 0.01-1.0 μg/ml,until reaching saturation at approximately 1 μg/mL or a meanfluorescence intensity (MFI) of 1000. In contrast, the staining of thenon-B lymphocyte population is detectable, but very low, and increasesslowly with increasing concentration of scFvIg.

TRU-015

CD20-specific SMIPs are prepared similarly. CD20-specific SMIPs aredescribed in co-owned US Patent Publications 2003/133939, 2003/0118592and 2005/0136049, each incorporated by reference herein in its entirety.An exemplary SMIP, TRU-015, is described below.

TRU-015 is a recombinant single chain protein that binds to the CD20antigen. The nucleotide and amino acid sequences of TRU-015 arerespectively set out in SEQ ID NOS: 3 and 4. The binding domain wasbased on a publicly available human CD20 antibody sequence. The bindingdomain is connected to the effector domain, the CH2 and CH3 domains ofhuman IgG1, through a modified CSS hinge region. TRU-015 exists as adimer in solution.

TRU-015 comprises the 2e12 leader peptide cloning sequence from aminoacids 1-23 of SEQ ID NO: 4; the 2H7 murine anti-human CD20 light chainvariable region with a lysine to serine (VHL11S) amino acid substitutionat residue 11 in the variable region, which is reflected at position 34in SEQ ID NO: 4; an asp-gly₃-ser-(gly₄ser)₂ linker, beginning at residue129 in SEQ ID NO: 4; the 2H7 murine anti-human CD20 heavy chain variableregion, which lacks a serine residue at the end of the heavy chainregion, i.e., changed from VTVSS to VTVS; a human IgG1 Fc domain,including a modified hinge region comprising a (CSS) sequence, and wildtype CH2 and CH3 domains.

Example 2

Culturing Host Cells with Carbohydrate Modifier

CHO cells transfected with TRU-016 or TRU-015 cDNA were cultured inshake flasks or wave bags with varying concentrations of variouscarbohydrate modifiers generally according to the procedures describedbelow.

For shake flask runs, log phase host cells were seeded in shake flasksat 100,000 cells/ml with carbohydrate modifier at the concentration tobe tested, and optionally with methotrexate (MTX) at 50 nM.

Cells were seeded at 3×10⁶/mL in 1350 mL of Ex-Cell 302 culture media(SATC Biosciences; with added non-essential amino acids, pyrucate,L-glutamine, pen/strep, HT Supplement and insulin, all from Invitrogen)at t=0 and brought to 5 L total volume at T>=72 hours. The cells wereincubated at 37° C. and 5% carbon dioxide and monitored for growth andviability daily starting at day 6-7. Supernatants were typicallyharvested at day 10-12 when cell viability dropped below 60%.

Sodium azide was added to 0.02%, cells were removed by centrifugationand supernatant was filter sterilized through a 0.22 uM filter. Someassays described in other examples herein were performed on thesupernatants as indicated, while other assays were performed on materialthat underwent further protein A purification. For wave bag runs, logphase host cells were seeded into 5 L wave bags at 100,000-200,000cells/ml in 10-20% conditioned Ex-Cell 302 media (SATC Biosciences; withadded non-essential amino acids, pyrucate, L-glutamine, pen/strep, HTSupplement and insulin, all from Invitrogen) with carbohydrate modifierat the concentration to be tested. Cells were incubated at 37° C. and 5%carbon dioxide and monitored daily for growth and viability.Supernatants were typically harvested at day 11-12 or when cellviability dropped below 50%.

Cells were removed by centrifugation in a Sorvall Legend at 3000 rpm(1932 rcf) for 20 minutes, the supernatant was filter sterilized. Someassays described in other examples herein were performed on thesupernatants as indicated, while other assays were performed on materialthat underwent further protein A purification.

TRU-016 produced by cells cultured with varying concentrations ofvarious carbohydrate modifiers is assayed for CD16 binding, ADCC, CDC,pharmacokinetic parameters and in vivo activity as described below.

FIGS. 1 and 2 are representative and show that treatment with thecarbohydrate modifier castanospermine at concentrations up to 1000 μMdid not affect cell counts or percent cell viability over all timeperiods sampled (up to 144 hours).

Example 3

Binding to FcRs

The immunoglycoproteins produced according to Example 2 were assayed invitro for binding to soluble Ig-fusion versions of Fcγ receptors, inwhich the extracellular domain of a receptor is fused to murine IgG2aFc.

The soluble Fcγ receptor materials were generated by fusing theextracellular domain of Fcγ Receptors I (Genbank Acc. No. BC032634), IIa(Genbank Acc. No. NM_(—)021642), IIb (Genbank Acc. No. BC031992), andIII-V158 (high affinity allele; Genbank Acc. No. X07934) and III-F158(low affinity allele), respectively, to a murine IgG2a Fc with a Pro toSer mutation at residue 238 (MIgG2aP238S). For both forms of Fcγ RIII(CD16), an HE4 leader was cloned onto CD16 amino acids 1-178 and thenfused to MIgG2aP238S.

The assays were carried out as follows. 500,000 WIL2-S cells (a Blymphoma cell line that expresses CD37 as well as CD20 on its surface)were incubated on ice in a Costar 96 well plate with 5 μg/ml of eitherTRU-015 or TRU-016 for 45 minutes in phosphate buffered saline (PBS)with 1% fetal bovine serum (FBS). Unbound TRU-015 or TRU-016 was removedby spinning the cells, washing with diluent (PBS+1% FBS) and spinningagain at 1200 rpm in a Sorvall Legend RT for 2 minutes. The cells werethen incubated with the desired FcγR-MIg fusion in the same diluent at aconcentration of 1 μg/ml on ice for 45 minutes.

The complexes (WIL2-S cells/SMIP/FcγR-MIg) were then incubated with PEconjugated AffiniPure F(Ab′)₂ Goat Anti-Mouse IgG (a mouse Fc-specificantibody with minimal cross reactivity with human Fc; JacksonImmunoresearch) at a 1:100 dilution. The cells were analyzed byone-color flow cytometry on a FACsCalibur using CellQuest software(Becton Dickinson).

If TRU-016 supernatants from Example 2 were used in this assay insteadof purified TRU-016 protein, the SMIP concentration in the supernatantwas quantified by direct staining of WIL2-S cells with dilutedsupernatant along with a TRU-016 standard. TRU-016 was detected bystaining with FITC conjugated F(Ab′)₂ Goat Anti-Human (gamma) [CaltagH10101] at a 1:50 dilution.

Binding to either the low affinity allele and high affinity allele weredetermined to correlate similarly to ADCC activity. An increase in CD16(low or high affinity allele) binding was correlated to an increase inADCC activity.

Representative results are displayed in FIGS. 3-6.

TRU-015 purified protein produced by CHO cells cultured in mediacontaining 0, 2, 5, 10, 30 or 100 μg/mL castanospermine was tested forCD16 binding (low affinity allele). Representative results of geometricmean fluorescence intensity are displayed in FIG. 3 and show adose-dependent increase in CD16 binding at increasing concentrations ofcastanospermine in the culture media.

TRU-016 supernatant produced by CHO cells cultured in media containing6,8a-diepicastanospermine at a concentration of 50 or 250 μM,swainsonine at a concentration of 50 or 250 μM, or deoxymannojirimycin(DMJ) at a concentration of 50 or 250 μM was tested for CD16 binding.Representative results of mean fluorescence intensity are displayed inFIG. 4 and show that both concentrations of DMJ increased CD16 binding.Although no effect was seen for 6,8a-diepicastanospermine or swainsonineat these concentrations, further tests with purified protein are carriedout to determine effect.

TRU-016 supernatant produced by CHO cells cultured in media containingkifunensine at a concentration of 0, 0.5, 1, 3, 5, or 10 μM was testedfor CD16 binding. Representative results of mean fluorescence intensityare displayed in FIG. 5 and show that kifunensine was much more potentthan DMJ at increasing CD16 binding and greatly increased CD16 bindingeven at the lowest concentration, 0.5 μM.

Protein A-purified TRU-016 produced by CHO cells cultured in mediacontaining 0, 10, 25, 50, 100 or 200 μM castanospermine was tested forCD16 binding. Representative results of mean fluorescence intensity aredisplayed in FIG. 6 and show a dose-dependent increase in CD16 bindingat increasing concentrations of castanospermine in the culture media.

Example 4

ADCC Activity

To determine the ADCC activity of purified TRU-016, labeled BJAB B cellswere used as targets and human peripheral blood mononuclear cells (PBMC)as effector cells. BJAB B cells (10⁷ cells) were labeled with 500 μCi/mL⁵¹Cr sodium chromate for 2 hours at 37° C. in IMDM/10% FBS. PBMCs wereisolated from heparinized, human whole blood by fractionation overLymphocyte Separation Media (LSM, ICN Biomedical) gradients. Reagentsamples were added to RPMI media with 10% FBS and serial dilutions ofeach reagent were prepared. The ⁵¹Cr labeled BJAB were added at 2×10⁴cells/well. The PBMCs were then added at 5×10⁵ cells/well for a finalratio of 25:1 effectors (PBMC):targets (BJAB). Reactions were set up inquadruplicate wells of a 96 well plate. Serial dilutions of TRU-016 wereadded to wells at a final concentration ranging from 10 ng/mL to 20μg/mL as indicated in the figures. Reactions were allowed to proceed for6 hours at 37° C. in 5% CO₂ prior to harvesting and counting. CPMreleased was measured on a Packard TopCounNXT from 50 μl dried culturesupernatant. Percent specific killing was calculated by subtracting (cpm[mean of quadruplicate samples] of sample−cpm spontaneous release)/(cpmmaximal release-cpm spontaneous release)×100, and data were plotted as %specific killing versus TRU-016 concentration. Representative resultsare displayed in FIGS. 7-10.

TRU-015 purified protein produced by CHO cells cultured in mediacontaining 0, 2, 5, 10, 30 or 100 μg/mL castanospermine was tested forADCC measured using PBMC from high affinity (V/V158) and low affinity(F/F158) CD16 donors. Representative results of % specific killing aredisplayed in FIGS. 7 and 8 (high affinity and low affinity donors,respectively) and show a dose-dependent increase in ADCC activity atincreasing concentrations of castanospermine in the culture media.

TRU-016 purified protein produced by CHO cells cultured in mediacontaining 0, 10, 25, 50, 100 or 200 μM castanospermine was tested forADCC. Representative results of % specific killing are displayed in FIG.9 and show a dose-dependent increase in ADCC activity at increasingconcentrations of castanospermine in the culture media.

TRU-016 purified protein produced by CHO cells cultured in mediacontaining 200 μM DMJ, 10 μM kifenunsine or 200 μM castanospermine wastested for ADCC. Representative results of % specific killing aredisplayed in FIG. 10 and show that all of these concentrations ofcarbohydrate modifiers improved ADCC of the immunoglycoproteins producedby the CHO cells.

Example 5

CDC Activity

To determine the CDC activity of TRU-016 purified protein producedaccording to Example 2, Ramos B cells were suspended in Iscoves(Gibco/Invitrogen, Grand Island, N.Y.) at 5×10⁵ cells/well in 75 μl.TRU-016 (75 μl) were added to the cells at twice the concentrationsindicated. Binding reactions were allowed to proceed for 45 minutesprior to centrifugation and washing in serum-free Iscoves. Cells wereresuspended in Iscoves with human serum (containing complement) atvarious concentrations. The cells were incubated 60 minutes at 37° C.Cells were washed by centrifugation and resuspended in staining mediawith 0.5 pg/ml propidium iodide. Samples were incubated 15 minutes atroom temperature in the dark prior to analysis by flow cytometry using aFACsCalibur and CellQuest software (Becton Dickinson).

TRU-015 purified protein produced by untreated CHO cells, or CHO cellstreated with 30 μg/ml castanospermine was tested for CDC activity.Results are displayed in FIG. 15.

TRU-016 purified protein produced by untreated CHO cells, or CHO cellscultured in media containing 200 μM DMJ, 10 μM kifenunsine or 200 μMcastanospermine, was tested for CDC activity. Results are displayed inFIG. 16.

These results show that CDC for carbohydrate-modified TRU-015 or TRU-016was similar to the CDC of corresponding protein produced by untreatedCHO cells, indicating that the presence of carbohydrate modifier in theculture medium of the host cells had no significant effect on CDC of theimmunoglycoprotein produced by the host cells.

Example 6

Pharmacokinetic Profile

Female BALB/c mice were injected i.v. with 200 μg of TRU-016 testprotein (TRU-016 produced by untreated CHO cells or by CHO cells treatedwith 200 μM DMJ, 10 μM kifenunsine or 200 μM castanospermine) at time 0.Serum samples were collected (3 mice per time point) at 15 min, 2, 6,24, 48, 72, 96, and 192 hours post injection.

The serum concentration of each TRU-016 test sample was determined in aFACS-based binding assay using the CD37+ Ramos human cell line. CD37+Ramos cells (5×10⁵ cells/well) were incubated in 96 well flat bottomplates along with the serum sample to be tested. Spiked serum sampleswere used for the standard curves. Cells were incubated at 4° C. for anhour and washed before addition of the detection antibody. Binding ofTRU-016 test protein to CD37+ Ramos cells was detected using afluorescein-conjugated goat anti-human IgG Fcγ fragment-specificantibody. Standard curves were used to construct a binding curve as afunction of antigen concentration. Briefly, standard curves consisted ofvarious known concentrations of the TRU-016 test protein spiked intonormal mouse serum diluted 1:20 in FACS buffer. The standard curves wererun in duplicate on each plate. Mean fluorescence intensities (MFI) fromthe FACS analysis were imported into Softmax Pro software and were usedto calculate serum concentrations of the TRU-016 test protein.

Results of the pharmacokinetic study showed that TRU-016 produced by CHOcells cultured in media containing 200 μM DMJ, 10 μM kifenunsine or 200μM castanospermine (displayed in FIG. 11) when administered to miceexhibited a pharmacokinetic profile similar to TRU-016 produced byuntreated CHO cells, indicating that carbohydrate modifier in theculture medium of the host cells had no significant effect on half-lifeor other pharmacokinetic parameters.

Repeating the CD16 assays on sera containing TRU-016 obtained from themice at 48, 72, 96 and 192 hours after administration of TRU-016 showedthat the sera retained its increased CD16 binding activity at all timepoints tested. Results are shown in FIG. 12.

Example 7

Carbohydrate-Modified Immunoglycoprotein Activity in vivo

Nude mice are administered 5×10⁶ Ramos cells subcutaneously on day 0 andinjected intravenously with 200 μg control human IgG or TRU-016 testprotein produced by CHO cells treated with 200 μM DMJ, 10 μM kifenunsineor 200 μM castanospermine on days 0, 2, 4, 6, and 8. Mice typicallydevelop tumors within 6 days and die shortly thereafter. Tumors aremeasured three times weekly with digital calipers and LabCat software,and tumor volume is calculated as ½[length×(width)]. Body weight is alsodetermined once a week.

Mice are sacrificed when the tumor reaches 1500 mm³ in size (1200 mm³ onFridays). Mice are also sacrificed if ulceration of a tumor occurs, thetumor inhibits the mobility of animal, or if weight loss equals orexceeds 20%.

Interim results for relative tumor volume at day 8 after the study wasinitiated are shown in FIG. 13. Data on % survival after the initiationof study are shown in FIG. 14 and below in Table 1.

TABLE 1 Median Survival Time Group (Days)* p value HuIgG 8 — CS TRU-01613 0.0054 DMJ TRU-016 13.5 0.0005 Kifu TRU-016 10 0.0084 *Values foreach of the carbohydrate-modified TRU-016 are significantly differentfrom that of the huIgG treated control group.

Results of this in vivo study showed that TRU-016 produced by CHO cellstreated with 200 μM DMJ, 10 ρM kifenunsine or 200 μM castanospermine wasable to reduce tumor volume and increase mean survival time in an animalmodel of cancer.

Example 8

Effect of Castanospermine at Varying Concentrations on ProteinProduction

Further experiments were performed to determine the effect ofcastanospermine concentration on cell viability, density and specificprotein production of TRU-016.

Prior to initiation of the experiments, DG44 CHO cells transfected withTRU-016 were grown in shake flasks in Ex-Cell™ 302 CHO serum-free media(SAFC Biosciences) supplemented with 1× non-essential amino acids(MediaTech), 1× sodium pyruvate (MediaTech), 4 mM L-glutamine(MediaTech), 500 nM methotrexate (MP Biomedicals) and 1 mg/L recombinantinsulin (Recombulin-GIBCO/Invitrogen Corp.) at 37° C. and 5% carbondioxide in a humidified incubator. A 200 mM stock concentration ofcastanospermine (Alexis Biochemicals) was prepared by dilution of thecastanospermine in sterile, distilled/deionized water (MediaTech) andfiltration through a 13 mm Acrodisc® with a 0.2 μm HT Tuffryn membrane(Pall Corporation). Stock solution was aliquoted into sterile, O-ringed,0.5 mL microcentrifuge tubes (Fisherbrand, Fisher Scientific) and frozenat −20° C. Approximately 1 hour prior to initiation of experiments,needed aliquots were thawed at room temperature and the contents of eachvial mixed well by vortexing.

For each experiment, cells in log phase growth were seeded in the abovemedium into a total volume of 60 mL in 250 mL shaker flasks at a densityof 200,000 cells/mL and CS added at the concentration to be tested.Final CS concentrations of 800 μM, 400 μM, 200 μM, 100 μM, 50 μM, 25 μMand 0 μM were each tested in duplicate flasks. All cultures wereincubated at 37° C. and 5% carbon dioxide in a humidified incubator andmonitored at least every other day for viable cell density and overallcell viability.

Cultures were harvested on day 8 when overall cell viability was 50-70%(Expt. 1) and 30-50% (Expt. 2). Cells and cellular debris were removedby centrifugation in a Sorvall Super T21 at 3000 rpm for 20 minutesafter which the supernatant was sterile filtered through a MilliporeSteriflip unit with a 0.22 μm Millipore Express Plus membrane and storedat 2-8° C. until purification.

Although cell viability and growth did not appear to be significantlyaffected as indicated by each sample's integral cell area (ICA), Table2, increasing concentrations of castanospermine appeared to reduceimmunoglycoprotein production. Results are shown in FIG. 17 and in Table2 below. Concentrations of 400 μm and 800 μm CS are shown to reduceTRU-016 protein production by approximately 40%-55% respectively.

TABLE 2 Average Viability TRU-016 ICA^(a) Specific CS Conc. at Produced10⁶ cells* Productivity^(b) (μM) Harvest (%) (ug/mL) ± SD days/mL(pg/cell/day) 800 70.6  99.65 ± 6.1 23.9 3.99 800 65.2 23.8 4.36 40068.2 124.93 ± 1.4 22.4 5.53 400 65.7 23.0 5.48 200 55.5 143.53 ± 1.421.9 6.60 200 51.1 22.0 6.47 100 54.4 161.83 ± 0.1 21.6 7.48 100 54.621.6 7.49 50 49.4 176.63 ± 1.0 21.6 8.15 50 50.0 21.4 8.27 25 53.9180.31 ± 6.6 21.4 8.22 25 54.5 21.1 8.78 0 65.1 208.24 ± 0.3 22.4 9.29 062.6 21.7 9.62 ^(a)Integral Cell Area (ICA) ICA = ((VCC_(n) +VCC_(n+1))/2) × (t_(n+1) − t_(n)) where VCC_(n) = viable cell density attime n VCC_(n+1) = viable cell density at time n + 1 units: 10⁶ cells *days/mL ^(b)Specific Productivity = total amount produced (ug/mL)/ICAunits: pg/cell/day

Example 9

Assay for Simultaneous Binding of TRU-016 to CD37 and FcγRIIIa (CD16)

Experiments were performed to determine the effect of castanospermineconcentration on functional activity of TRU-016 as measured by itsbinding to FcγRIIIa and its binding to target antigen CD37.

TRU-016 produced as described in Example 8 was tested in the followingassay, which simultaneously evaluates the ability of the TRU-016 bindingdomain to bind to a CD37 expressing target cell and the ability of theFc portion of the TRU-016 SMIP to bind a fusion protein of human CD16and murine IgG Fc.

The target cell utilized is the Daudi (ATCC CRL-213) cell line. Daudicells are a human B-lymphoblastoid cell line derived from a Burkitt'slymphoma and express high levels of CD37. The custom solubleCD16:MuIgGFc fusion protein is human CD16 (low affinity polymorphism)linked to a murine IgG Fc.

The appropriate number of Daudi cells (350,000/well times the number ofwells) is aliquoted and centrifuged at 250×g for 5 minutes at 15° C. Thesupernatant is removed. One percent cold paraformaldehyde is prepared bydiluting the 4% stock from USB (USB US19943) 1:4 with FACS Buffer. FACSBuffer is prepared by adding 2% FBS (Gibco) to Dulbecco's PBS(Invitrogen) (v/v) and sterile filtering with a 0.22 μm filter. FACSBuffer is stored and used at 4° C. The cells are resuspended in 1%paraformaldehyde (a volume equal to 50 μL/well times the number ofwells) and plated out in a round bottom 96-well plate. The cells areincubated for 30 minutes at 4° C. Following this incubation the cellsare washed by adding 150 μL of FACS Buffer to each well, centrifuging at250×g for 3 minutes at 15° C. and the supernatant removed. The cells areresuspended in 50 μL of FACS Buffer. TRU-016 is diluted in FACS Buffer,at concentrations ranging from saturation to background levels (24μg/mL-0.011 μg/mL), added to the appropriate wells, 50 μL/well, and thecells incubated for 25 minutes at 4° C. The CD16:MuIgGFc fusion proteinis diluted in FACS Buffer to a saturating level (20 μg/ml) and added tothe assay (50 μL/well) and incubated for an additional 30 minutes at 4°C. to form a complex with the TRU-016 that has bound to the cellsurface. Any unbound reagents are removed from the well by centrifugingat 250×g for 3 minutes at 15° C., removing the supernatant and thenwashing 3 times with 200 μL/well of FACS Buffer. The cells are thenincubated with a fluorophore (R-phycoerythrin, Jackson 115-116-071)tagged F(ab′)2 antibody, specific to murine Fc (and selected to beminimally reactive to human Fc). This antibody will bind to the MuIgGFcportion of the CD16:MuIgGFc fusion protein. The antibody is diluted1:200 in FACS Buffer and 100 μL is added to each well. The plate isincubated at 4° C. in the dark for 45 minutes. Any unbound R-PE isremoved by adding 150 μL of FACS Buffer to each well and centrifuging at250×g for 3 minutes at 15° C. followed by removal of supernatant. Thisis followed by a second wash with 200 μL/well FACS Buffer, centrifugingat 250×g for 3 minutes at 15° C. and removal of supernatant. The cellsare resusupended with 200 μL/well 1% paraformaldehyde and stored at 4°C. overnight.

Each sample's bound fluorescence is measured on a BD FACSCalibur flowcytometry system and analyzed with Cell Quest Pro software (BectonDickinson, ver 5.2). The GeoMean fluorescence intensity for each sampleis plotted relative to the TRU-016 concentration. A dose response isgenerated and fit to a 4-parameter logistic (4-PL) curve using SoftMaxPro software (Molecular Devices, ver 5.0.1). Titrations of TRU-016 areutilized to create a dose response curve of test and reference materialfor comparison. The “D”-parameter (Maximal curve asymptote) is used asreference for comparison of treated and untreated samples. An increasein the “D” value represents an increase in the binding activity for thecorresponding sample.

Results of the experiment are displayed in FIG. 18 and show adose-dependent binding response relative to concentration of CS up to400 μM, at which point the binding appears to level off.

To demonstrate that the enhanced binding of CS treated TRU-016 samplesto CD16 was not in part due to enhanced binding of the molecules toCD37, the above assay was repeated except that after addition andincubation of treated or untreated TRU-016 samples in the assay plate,unbound TRU-016 is removed from the well by centrifuging at 250×g for 3minutes at 15 oC, removing the supernatant and then washing 3 times with200 μL/well of FACS buffer. The cells are then incubated with aFITC-conjugated goat anti-human IgG Fc specific antibody (CaltagH10501). This antibody will bind to the Fc region of the human IgG chainof TRU-016 bound to the cells. The antibody is diluted 1:50 in FACSbuffer and 100 μL is added to each well. The plate is incubated at 4 oCin the dark for 45 minutes. Any unbound FITC-labeled antibody is removedby adding 100 μL of FACS buffer to each well, centrifuging at 250×g for3 minutes at 15 oC followed by removal of supernatant. This is followedby a second wash with 200 μL/well FACS buffer. The cells areresusupended with 200 μL/well 2% paraformaldehyde and stored at 4 oCovernight. Each sample's bound fluorescence is measured on a BDFACSCalibur flow cytometry system and analyzed using Cell Quest Prosoftware (Becton Dickinson, ver 5.2). The GeoMean fluorescence intensityfor each sample is plotted relative to the TRU-016 concentration. A doseresponse curve is generated and fit to a 4-parameter logistic (4-PL)curve using the SoftMax Pro software (Molecular Devices, ver 5.0.1).Titrations of TRU-016 are utilized to create a dose response curve ofthe untreated control and CS treated samples for comparison.

As shown in FIG. 19, the dose response binding curves to CD37 expressingcells for all CS treated samples were essentially identical to eachother and to the untreated TRU-016 sample, indicating that treatmentwith CS did not alter the binding of TRU-016 to its specific targetantigen.

Example 10

Antibody Dependent Cellular Cytotoxicity (ADCC) Assay

Experiments were performed to determine the effect of castanospermineconcentration on functional activity of TRU-016 as measured by ADCCactivity.

TRU-016 produced as described in Example 8 is incubated with theCD37-expressing Daudi cancer B-cell line in conjunction with primaryhuman peripheral blood lymphocytes (PBL's) effector cells to assess ADCCactivity.

Daudi target cells (5×10⁶) are added to a 15 ml conical tube and thencentrifuged at 250×g for 5 minutes at 20° C. and the supernatantremoved. The cell pellet is resuspended by the addition of 0.3 mCiChromium-51 (⁵¹Cr, GE Healthcare, CJ51). The cells are incubated for 75minutes at 37° C. with 5% CO₂, allowing the cells to incorporate theradioactive isotope. The cells are then washed three times to remove anyunincorporated ⁵¹Cr. This is done by adding 10 mL of complete media—IMDM(Gibco) with 10% FBS (Gibco)—to the tube, centrifuging at 250×g for 5minutes at 20° C. followed by removal of supernatant. The finalresuspension is in 11.5 mL of complete media. TRU-016 is diluted incomplete media, at concentrations that are able to generate maximal tobackground levels of cell lysis (500 ng/mL-0.005 ng/mL). Thesetitrations are plated out, 50 μL/well, in a round bottom 96 well plate.The ⁵¹Cr labeled target cells are added to the dose titrations ofTRU-016 at 50 μL/well and the control wells (control media withoutTRU-016). PBL's are isolated from fresh heparinized whole blood bydensity gradient centrifugation using Lymphocyte Separation Media as perprotocol (LSM, MP Biomedical, 50494/36427). PBL effector cells areadded, 100 μL/well, to the wells at a ratio of between 25:1-30:1(effector:target). The assay is incubated for 4.5-5 hours at 37° C., 5%CO₂. The effector cells lyse the target cells relative to the TRU-016concentration, releasing a proportional amount of ⁵¹Cr into the assaysupernatant. Following the incubation the plate is centrifuged at 250×gfor 3 minutes at 20° C. A 25 μL volume of cell-free supernatant isremoved from all wells to a scintillation plate (Perkin Elmer 6005185)and dried overnight. The amount of ⁵¹Cr isotope in each well of thescintillation plate is measured using a Topcount plate reader (PerkinElmer, C9904VO). The data are expressed as percent of specific release.Specific release is calculated as:(Sample value−Spontaneous value)/(Maximum value−Spontaneous value)*100%

-   -   Spontaneous=amount of ⁵¹Cr released from target cell only    -   Maximum release=amount of ⁵¹Cr released from targets treated        with detergent lysing agent    -   Background Control=amount of ⁵¹Cr released from target        cells+effector cells (No TRU-016)

A dose response is generated and fit to a 4-parameter logistic curveusing SoftMax Pro software (Molecular Devices, ver 5.0.1). Titrations ofTRU-016 are utilized to create dose response curves of test andreference material for comparison. The EC50 values for the treatedarticles are compared to the untreated control (no CS) to determine thepercent increase in ADCC activity. Table 3 below summarizes the datadisplayed in FIG. 20. The data indicate that the ADCC activity ofTRU-016, treated with CS over a range of 100 μM-800 μM finalconcentration, is significantly increased relative to untreated TRU-016.

TABLE 3 Donor AF Donor AF Donor Donor Hetero- Hetero- Q High N Lowzygous zygous Sample ID 1:17 1:17 1:25 1:13 Control 1.24 2.60 0.23 0.37CS 0 μM CS 100 μM 0.25 (502%) 0.70 (370%) 0.03 (728%) n/a CS 200 μM 0.21(589%) 0.54 (479%) n/a 0.06 (579%) CS 400 μM 0.24 (515%) 0.53 (492%) n/a0.08 (440%) CS 800 μM 0.25 (492%) 0.63 (414%) n/a 0.08 (451%) Ratio 1:X= Target to Effector (PBMC freshly isolated from whole blood) Donors arehomozygous high affinity (High), homozygous low affinity (Low), orHeterozygous for CD16 allele.

Example 11

Effect of Castanospermine at Varying Concentrations on CHO-K1 TRU-016Protein Production

Further experiments were performed to determine the effect ofcastanospermine concentration on cell viability, density and specificprotein production of TRU-016 in, an alternate cell line (CHO-K1)producing TRU-016.

Prior to initiation of the experiments, CHO-K1 cells transfected withTRU-016 (CAS029f053) were grown in vented shake flasks in CD-CHOchemically-defined media (GIBCO-Invitrogen) supplemented with 25 uMmethionine sulfoximine (MSX) at 37° C. and 5% carbon dioxide in ahumidified incubator. Shaker speed was set at 125 RPM. A 400 mM stockconcentration of castanospermine (Alexis Biochemicals) was prepared bydilution of the CS in sterile, distilled/deionized water (MediaTech)followed by filtration through a 13 mm Acrodisc® with a 0.2 μm HTTuffryn membrane (Pall Corporation). Stock solution was aliquoted intosterile, O-ringed, 0.5 mL microcentrifuge tubes (Fisherbrand, FisherScientific) and frozen at −20° C. Approximately 1 hour prior toinitiation of experiments, needed aliquots were thawed at roomtemperature and the contents of each vial mixed well by vortexing.

For the experiment, cells in log phase growth were seeded in the abovemedium into a volume of 60 mL in 250 mL shaker flasks at a density of220,000 cells/mL and CS added at the concentration to be tested. FinalCS concentrations of 800 μM, 400 μM, 200 μM, 100 μM, 50 μM, 25 μM and 0μM were each tested in duplicate flasks. All shake flasks were incubatedon a shaker platform set at 125 RPM at 37° C. and 5% carbon dioxide in ahumidified incubator. Cultures were monitored at least every other dayfor viable cell density and overall cell viability.

Cultures were harvested on day 10 when overall cell viability was49-62%. Cells and cellular debris were removed by centrifugation in aSorvall Super T21 at 3000 rpm for 20 minutes after which the supernatantwas sterile filtered through a Millipore Steriflip unit with a 0.22 μmMillipore Express Plus membrane and stored at 4° C. until purified.

Inclusion of castanospermine in the cultures in the final concentrationrange of 25-800 μM did not appear to significantly affect, positively ornegatively, overall cell viability or growth as indicated by each testsample's integral cell area (ICA) compared to that of the controlsamples (Table 4). Interestingly, the presence of castanospermine hadonly a very weak suppressive effect on the production of TRU-016 with nomore than a 7.5% decrease relative to control noted (Table 4), even atthe highest concentration of 800 μM. In fact, the maximal suppressiveeffect of CS on TRU-016 production was reached at 200 μM and remainedessentially the same at higher concentrations of CS. Average specificproductivity of the cells at each concentration of CS was essentiallythe same and very similar to that observed with the control cultures(Table 4) suggesting that CS had no significant effect on this property.

TABLE 4 Viability Average AverageTRU- Average Average at TRU-016 016Produced ICA (10⁶ Specific Flask Test Harvest Produced as a % of cells*Productivity # Condition (%) (μg/mL) ± SD Control ± SD days/mL) ± SD^(a)(pg/cell/day) ± SD^(b) 275 CS - 800 μM 58.1 401.14 ± 4.1  92.5 ± 1.037.6 ± 1.0 10.68 ± 0.40 276 CS - 800 μM 59.7 277 CS - 400 μM 56.1 401.22± 13.3 92.6 ± 3.1 37.6 ± 0.3 10.68 ± 0.28 278 CS - 400 μM 62.4 279 CS -200 μM 61.8 401.93 ± 5.0  92.7 ± 1.2 40.1 ± 2.3 10.04 ± 0.69 280 CS -200 μM 56.4 281 CS - 100 μM 61.2 408.60 ± 6.9  94.3 ± 1.6 39.0 ± 0.410.48 ± 0.07 282 CS - 100 μM 55.1 283 CS - 50 μM 49.4 422.84 ± 27.8 97.5± 6.4 41.6 ± 0.9 10.18 ± 0.45 284 CS - 50 μM 61.3 285 CS - 25 μM 57.7419.36 ± 11.1 96.7 ± 2.6 40.8 ± 0.4 10.29 ± 0.18 286 CS - 25 μM 53.0 289CS - 0 μM 56.7 433.50 ± 10.2 100.0 ± 2.4  40.4 ± 0.0 10.73 ± 0.24 290CS - 0 μM 57.3 ^(a)Integral Cell Area (ICA) = ((VCC_(n) + VCC_(n+1))/2)× (t_(n+1) − t_(n)) where VCC_(n) = viable cell density at time n andVCC_(n+1) = viable cell density at time n + 1. Units = 10⁶ cells *days/mL ^(b)Specific Productivity = total amount produced (ug/mL)/ICA.Units = pg/cell/day

Example 12

Assay for Simultaneous Binding of CHO-K1 Produced TRU-016 and TRU-016Glycovariants to CD37 and FcγRIIIa (CD16)

An experiment was performed to determine the effect of castanosperminemodification of the N-linked oligosaccharide on TRU-016 with regard tothe functional activity of the molecule as measured by its binding toFcγRIIIa (CD16) and its binding to target antigen CD37. The experimentwas performed as detailed in Example 9 with each of the CS treatedsamples and controls described in Example 11 and the results graphicallyrepresented in FIG. 21. As shown in FIG. 21, CS induced a dose-dependentimprovement in TRU-016 glycovariant binding to CD16 that became maximal(approximately 7-fold) at CS concentrations of 100 μM or greater.

To demonstrate that the enhanced binding of CS induced TRU-016glycovariant samples to CD16 was not in part due to enhanced binding ofthe molecules to CD37, each of the different CS treated samples werecompared to the control TRU-016 sample in the CD37 binding assaydescribed in Example 9. As shown in FIG. 22, the dose response bindingcurves of TRU-016 glycovariants to CD37 expressing cells were virtuallyidentical to each other and to that of the control TRU-016 sample,indicating that culture with CS did not alter the binding of TRU-016 toits specific target antigen.

Example 13

Antibody Dependent Cellular Cytotoxicity (ADCC) Assay with CHO-K1Produced TRU-016 Control and Glycovariant Samples

Experiments were performed to determine the effect of increasingconcentration of CS in the culture media on the functional activity ofresulting TRU-016 as measured by its ability to potentiate ADCC. Thisassay was carried out as described in Example 10 although in this case,PBL effector cells from each of the three potential CD16 genotypes [158Phe/158 Phe (F/F—homozygous low binder), 158 Phe/158 Val(F/V—heterozygous binder) and 158 Val/158 Val (V/V—homozygous highbinder)] were tested. As shown in FIG. 23 and Table 5, samples ofTRU-016 produced in the presence of 400 μM, 100 μM and 50 μM CS wereessentially equally active in enhancing ADCC compared to the untreatedcontrol. Furthermore, this observation was consistent regardless of thegenotype of the PBL effector cell donor. The fold improvement in ADCCcapacity, as determined by EC₅₀ comparison of the glycovariant samplesversus control, appeared to be greatest in the case of the homozygoushigh binder (V/V) donor (maximum of 27.6-fold) compared to theheterozygous (F/V) (maximum 12-fold) and homozygous low binder (F/F)(maximum 13.8-fold) donors. In addition, with all three PBL donor types,there was observed a slightly higher level of target cell cytotoxity(2-8%) with the TRU-016 glycovariant molecules as compared to TRU-016cultured in the absence of CS. This increased level of target cellcytotoxicity was most notable with the heterozygous donor's effectorcells.

TABLE 5 V/V (high) F/V (low/high) F/F (low) Donor Donor Donor CS FoldFold Fold Treat- EC₅₀ Improve- EC₅₀ Improve- EC₅₀ Improve- ment (ng/mL)ment (ng/mL) ment (ng/mL) ment 400 μM 0.05 27.6 0.61 10.4 0.26 12.6 100μM 0.06 20.4 0.53 12.0 0.24 13.8  50 μM 0.08 14.8 0.62 10.3 0.27 12.0None 1.24 1.0 6.31 1.0 3.25 1.0

Example 14

Effect of Castanospermine at Varying Concentrations on ProteinProduction in Bioreactors

Prior to growth in bioreactors, DG44 CHO cells transfected with TRU-016were grown in the absence of castanospermine in vented shake flasks asdescribed in Example 8.

For the bioreactor run, 3 L Applikon bioreactors were used. Growth mediafor cells was as described in Example 8 except that there was nomethotrexate present. Starting culture volume for each bioreactor was1700 mLs with a starting density of 3.5×10⁵ cells/mL. A total of eightbioreactors were run in pairs with CS at 0 μM, 300 μM, 350 μM and 400 μMfinal concentration (one of the 400 μM CS bioreactors was lost tocontamination on day 2 of culture). Castanospermine was added at theinitiation of culture in the bioreactors with no further addition duringthe culture period. Other culture conditions included a temperature setpoint of 37° C., pH set point of 6.9 and dissolved O₂ set point of 50%.Bioreactors were run in a fed-batch mode with L-glutamine and glucoseadded on an as needed basis. Soy hydrolysate (CHO Feed BioreactorSupplement, C1615, Sigma) was added at 1% of starting culture volume ondays 2, 4 and 6. Bioreactors were sampled daily for viable cell density,percent viable cells, media chemistries and osmolality. All cultureswere harvested on day 8 when overall cell viability ranged from 39-50%.

Cell growth and viability in the bioreactors was quite comparable asshown for integral cell area (ICA) of the cultures in Table 6 and thefact that they were all harvested at the same time. Production ofTRU-016, however, was somewhat inhibited in all bioreactors containingCS in the culture medium as compared to the control, no CS, bioreactors(Table 7). Suppression of TRU-016 production ranged from 24.9-32% of thenon-treated control bioreactors, somewhat less than the 35-40% thatmight have been predicted from shake flask experiments. As the ICAs ofthese bioreactors were quite similar, the primary explanation for lossof TRU-016 production may be a decreased specific productivity of thecell line when cultured in the presence of castanospermine (Table 6) aswas seen in shake flask experiments.

TABLE 6 Viability TRU-016 at TRU-016 Produced ICA (10⁶ Specific CSHarvest Produced as a % of cells* Productivity Bioreactor Concentration(%) (μg/mL) Control^(a) days/mL)^(b) (pg/cell/day)^(c) R093 CS - 0 μM41.0 434.1 101.5 50.0 8.69 R094 CS - 0 μM 48.2 421.3 98.5 47.0 8.97 R097CS - 300 μM 42.4 321.3 75.1 46.2 6.96 R098 CS - 300 μM 48.8 291.0 68.047.5 6.12 R095 CS - 350 μM 39.0 314.7 73.6 46.6 6.75 R096 CS - 350 μM41.6 318.1 74.4 45.5 7.00 R099 CS - 400 μM 50.4 311.0 72.7 47.9 6.49^(a)Based on an average (427.7 ug/mL) of TRU-016 produced in R093 andR094 ^(b)Integral Cell Area (ICA) = ((VCC_(n) + VCC_(n+1))/2) × (t_(n+1)− t_(n)) where VCC_(n) = viable cell density at time n and VCC_(n+1) =viable cell density at time n + 1. Units = 10⁶ cells * days/mL^(c)Specific Productivity = total amount produced (ug/mL)/ICA. Units =pg/cell/day

Example 15

Assay for Simultaneous Binding of DG44 CHO Produced TRU-016 and TRU-016Glycovariants to CD37 and FcγRIIIa (CD16)—Bioreactor Generated TRU-016

TRU-016 control and glycovariant proteins were purified from thesupernatants of all bioreactors described in Example 14 and evaluated asdescribed in Example 9 for their ability to simultaneously bind to thespecific target antigen CD37 and to FcγRIIIa to determine what effectproduction of TRU-016 in the presence of varying concentrations ofcastanospermine had on these aspects of TRU-016 functional activity.

As shown in FIG. 24, TRU-016 glycovariants purified from bioreactorscontaining CS supplemented media at all three CS concentrations stated,displayed nearly identical binding curves over the concentration rangetested and that all these samples demonstrated significantly enhancedbinding, at least 4-5 fold, compared to the samples from the control, noCS reactors.

Example 16

Antibody Dependent Cellular Cytotoxicity (ADCC) Assay—BioreactorGenerated DG44 CHO Produced TRU-016 and TRU-016 Glycovariants

Experiments were performed to determine the effect of increasingconcentration of castanospermine in the culture media of DG44 CHOproduced TRU-016 on the functional activity of resulting TRU-016 asmeasured by its ability to potentiate ADCC.

In addition to purification of TRU-016 from all bioreactors described inExample 14 via the abbreviated protein A scheme as detailed in Example 1(termed PA samples), TRU-016 and TRU-016 glycovariant was purified frombioreactors R094 (no CS) and R099 (400 μM CS), respectively. This methodincluded a 2 column process involving Protein A capture followed by aviral inactivation step at low pH and then a ceramic hydroxyapatite(CHT) polishing step. The samples from this purification method arereferred to as CHT samples.

Comparison of the two PA and CHT purified preparations from the 0 and400 μM CS containing bioreactors for their ability to potentiate ADCCassay as described in Example 10 showed that the method of purificationmade little difference in how the respective samples performed in theassay (FIG. 25) and that inclusion of 400 μM CS in the culture media forDG44 CHO produced TRU-016 yielded a TRU-016 glycovariant that was12-fold (PA sample) to 18-fold (CHT sample) more potent for ADCCcompared to like-purified TRU-016 produced in the absence of CS.

Example 17

Analytical Characterization of TRU-016 Control and Glycovariants usingLiquid Chromatography Coupled to Mass Spectrometry (LCMS) Whole MassAnalysis

LCMS analysis was used to confirm the peptide mass and to monitorchanges in glycan distribution of TRU-016 immunoglycoprotein generatedin the absence (TRU-016) and presence (TRU-016 glycovariant) of varyingconcentrations of CS. TRU-016 and glycovariants thereof exist as a dimerunder non-reducing conditions. Therefore, to simplify the analysis ofthe heterogeneous mixture of glycoforms, analysis was performed on thereduced, monomeric species. Prior to analysis, the molecule was reducedwith 20 mM DTT in 4.8M guanidine. Twenty pmol of monomeric protein wasthen injected onto a POROS R1 10 μm column and eluted with acetonitrileinto an ESI-TOF (Agilent) mass spectrometer detector.

Deglycosylation prior to LCMS analysis by treatment withpeptide-N-glycosidase F (PNGaseF) was used to confirm the parent peptidespecies. The resulting mass spectra were then deconvoluted and theglycan species identified by subtraction of the protein mass. Resultingmasses were analyzed using GlycoMod to correlate to known glycoforms,e.g., the observed mass of 53630.7 Daltons (Da) corresponds to thetheoretical mass of the native peptide (˜52414 Da) plus that of theknown glycan structure Hex₂+(Man)₃(GlcNAc)₂ (˜1216 Da), to within 0.2Da, or 4 PPM.

The relative abundance of individual glycopeptide species was thenestimated by comparing intensities of the deconvoluted peaks of eachspecies identified. The CS-dependent shift in glycoforms was thenmonitored by comparing the relative abundance of these deconvoluted massspecies as a function of CS concentration.

This type of analysis was performed on purified TRU-016 control andglycovariants produced by DG44 CHO cells as well as CHO-K1 cells and thedata are summarized in Tables 7 and 8, respectively. Based on thisanalysis, the observed mass species of the TRU-016 control produced inboth CHO cell lines were consistent with the expected amino acidsequence with a typical, heterogeneous mammalian glycosylation pattern.The observed glycoforms were dominated by two glycans,(Hex)₁(HexNAc)₂(Deoxyhexose)₁+(Man)₃(GlcNAc)₂ and(HexNAc)₂(Deoxyhexose)₁+(Man)₃(GlcNAc)₂, which correlate to the G1F andG0F N-linked glycans, respectively. Non-fucosylated, oligomannoseglycoforms represent only a small proportion (DG44 CHO produced, Table 7or CHO-K1 produced, Table 8) of the observed glycans.

Upon production in culture media containing castanospermine, theobserved distribution of glycan species on resulting TRU-016glycovariants changed in a CS dose-dependent manner to predominantlyhigh-hexose type glycoforms dominated by a (Hex₇)+(Man)₃(GlcNAc)₂species. Thus, with increasing concentration of CS, the proportion ofcomplex, fucosylated glycoforms decreased with a concomitant increase inthe proportion of high-hexose glycans.

TABLE 7 Relative Proportion of Glycoform Type Glycoform None 200 μM 300μM 400 μM 500 μM Glycoform Composition Type Control CS CS CS CSNon-Glycosylated Ø 5.9 9.0 9.1 9.0 7.7 (Hex)₂(HexNAc)₂(DeoxyHex)₁ +(Man)₃(GlcNAc)₂ Complex 3.5  —^(a) — — — G2F(Hex)₁(HexNAc)₂(DeoxyHex)₁ + (Man)₃(GlcNAc)₂ Complex 30.9 8.1 6.0 3.43.7 G1F (HexNAc)₂(DeoxyHex)₁ + (Man)₃(GlcNAc)₂ Complex 41.4 17.8 6.3 3.33.9 G0F (Hex)₂ + (Man)₃(GlcNAc)₂ High 3.2 0.6 — — — hexose (Hex)₃ +(Man)₃(GlcNAc)₂ High 4.3 — — — — hexose (Hex)₄ + (Man)₃(GlcNAc)₂ High4.4 — — — — hexose (Hex)₅ + (Man)₃(GlcNAc)₂ High 5.0 15.9 14.5 12.5 10.7hexose (Hex)₆ + (Man)₃(GlcNAc)₂ High 1.5 — — — — hexose (Hex)₇ +(Man)₃(GlcNAc)₂ High — 46.0 57.9 65.7 68.5 hexose (Hex)₈ +(Man)₃(GlcNAc)₂ High — 1.1 4.2 4.6 4.2 hexose (Hex)₉ + (Man)₃(GlcNAc)₂High — 1.6 1.9 1.6 1.3 hexose ^(a)Not detected

TABLE 8 Relative Proportion of Glycoform Type^(a) Glycoform None 25 μM50 μM 100 μM 200 μM 400 μM 800 μM Glycoform Composition Type Control CSCS CS CS CS CS Non-Glycosylated Ø 0.8 —^(b) 0.8 — 1.3 0.9 1.0(Hex)₁(HexNAc)₂(DeoxyHex)₁ + (Man)₃(GlcNAc)₂ Complex 17.1 11.7 7.4 4.00.9 — — G1F (HexNAc)₂(DeoxyHex)₁ + (Man)₃(GlcNAc)₂ Complex 75.9 53.328.5 17.1 5.6 3.0 1.7 G0F (HexNAc)₂ + (Man)₃(GlcNAc)₂ Complex 2.9 1.7 —— — — — G0 (Hex)₅ + (Man)₃(GlcNAc)₂ High — 13.3 16.3 13.3 8.5 5.2 5.3hexose (Hex)₇ + (Man)₃(GlcNAc)₂ High — 19.8 46.8 63.2 81.9 87.7 87.8hexose ^(a)Average of results from duplicate cultures ^(b)Not detected

Example 18

LCMS Tryptic Digest Glycoprofiling of TRU-016/CS-Generated Glycovariants

The purpose of this study was to determine semi-quantitativeglycoprofiles for the glycopeptides in control and glycovariant lots ofTRU-016. This study identifies the single peptide of TRU-016 whichcontains carbohydrate, shows the consistency of that glycosylatedpeptide between control and glycovariant products and illustrates theshift in glycoform that is associated with that single glycopeptide(containing the N-linked glycosylation site). LCMS of a tryptic digestwas used to identify the N-linked glycosylation site and attachedglycoforms, as a function of CS concentration in cell culture, asdescribed below:

Samples analyzed: TRU-016 control; Glycovariant TRU-016 200 μM CS;Glycovariant TRU-016 600 μM CS.

Protein samples were reduced using a 6 M Guanidine HCl, 0.002 M EDTA,0.02 M Tris denaturing solution, pH 8.33. Five microliters of 1M DTT wasadded to make a final concentration of 5 mM and the sample incubated at37° C. for one hour.

Following reduction, the sample was alkylated with 10 mM iodoacetamide.The sample was incubated for one hour while rocking at ambienttemperature, transferred into a dialysis cassette and dialyzed overnightagainst 50 mM ammonium bicarbonate, pH 8.28. The samples were removedfrom the dialysis buffer and divided into 2 equal aliquots at 62.5 μgeach. Reduced and alkylated protein was stored at −20° C. until needed.

One vial of each sample was removed and thawed at room temperature. Toeach vial, 1:12.5, w:w of trypsin at 0.5 mg/ml was added. Samples wereincubated at 37° C. for 18 hours. The samples were transferred to HPLCvials and placed in the autosampler.

Mass spectrometry data was collected on a Q-TOF Ultima mass spectrometer(Micromass/Waters) using electrospray ionization (ESI) in positive ionmode. Data was acquired from m/z 200-1950 in MS mode. Prior to analysis,the mass spectrometer was calibrated using a 5th order fit on fragmentions of Glu-Fibrinopeptide covering a range from m/z 175 to 1285.

The results of the analysis show that Asn333 (corresponds to Asn297 inKabat numbering) is the only potential site of N-linked glycosylation inTRU-016. Detailed analysis of all observed mass species in the trypticdigest generated no evidence of O-linked glycosylation.

Tryptic map chromatograms for the test articles are shown in FIG. 26.The chromatograms were extracted for ions between m/z 600-1800 (the m/zrange for the known N-linked tryptic glycopeptide). All ions found inthis range were examined more closely to determine if these masses werepotentially glycopeptides. The known mass of the peptide that containsAsn333 was subtracted from the observed masses and the resulting massesthen analyzed using GlycoMod to correlate to known glycoform structures.

All observed glycopeptide masses were found to elute within 1 minute ofeach other. Once a potential candidate was identified, the ion abundanceas determined by peak height was compiled to provide relative quantitiesof glycoforms. This data is summarized below. The sample which resultsfrom treatment with 600 μM CS is predominantly modified with high hexosetype glycoforms, with the predominant species containing ten hexoseunits. The 200 μM CS sample was found to contain a distribution ofglycoforms intermediate between the 600 μM CS sample and glycovariantcontrol.

Chromatogram and extracted glycopeptide mass chromatograms of TRU-016tryptic digest: The TRU-016 control consistently exhibits a profile witha glycopeptide mass species at ˜30.6 minutes RT. The 600 μM CS-treatedglycovariant exhibits a glycopeptide peak fully shifted to 30 minutes,while the 200 μM CS sample exhibits a mixture of peaks that elutebetween 30 and 30.6 minutes.

The tables below show relative abundance of glycoforms identified in theglycopeptide peaks illustrated above and the corresponding glycoformcompositions identified by GlycoMod.

N-linked profile ASN³³³ for TRU-016 control Relative GlycoformComposition Type abundance (Hex)₁ + (Man)₃(GlcNAc)₂ high mannose 0.6%(Hex)₂ + (Man)₃(GlcNAc)₂ high mannose 1.6% (HexNAc)₁ (Deoxyhexose)₁ +hybrid/complex 0.9% (Man)₃(GlcNAc)₂ (Hex)₃ + (Man)₃(GlcNAc)₂ highmannose 2.1% (Hex)₁ (HexNAc)₁ (Deoxyhexose)₁ + hybrid/complex 0.2%(Man)₃(GlcNAc)₂ (HexNAc)₂ (Deoxyhexose)₁ + hybrid/complex 58.9%(Man)₃(GlcNAc)₂ (Hex)₄ + (Man)₃(GlcNAc)₂ high mannose 1.5% (Hex)₁(HexNAc)₂ (Deoxyhexose)₁ + hybrid/complex 31.2% (Man)₃(GlcNAc)₂ (Hex)₅ +(Man)₃(GlcNAc)₂ high mannose 0.9% (Hex)₂ (HexNAc)₂ (Deoxyhexose)₁ +hybrid/complex 1.3% (Man)₃(GlcNAc)₂ (Hex)₆ + (Man)₃(GlcNAc)₂ highmannose 0.7%

N-linked profile ASN³³³ for Glycovariant (200 μM CS) Relative GlycoformComposition Type abundance (Hex)₁ + (Man)₃(GlcNAc)₂ high mannose 0.8%(Hex)₂ + (Man)₃(GlcNAc)₂ high mannose 2.3% (HexNAc)₁ (Deoxyhexose)₁ +hybrid/complex 0.9% (Man)₃(GlcNAc)₂ (Hex)₃ + (Man)₃(GlcNAc)₂ highmannose 1.5% (Hex)₁ (HexNAc)₁ (Deoxyhexose)₁ + hybrid/complex 0.6%(Man)₃(GlcNAc)₂ (HexNAc)₂ (Deoxyhexose)₁ + hybrid/complex 36.0%(Man)₃(GlcNAc)₂ (Hex)₄ + (Man)₃(GlcNAc)₂ high mannose 2.4% (Hex)₁(HexNAc)₂ (Deoxyhexose)₁ + hybrid/complex 24.9% (Man)₃(GlcNAc)₂ (Hex)₅ +(Man)₃(GlcNAc)₂ high mannose 10.9% (Hex)₂ (HexNAc)₂ (Deoxyhexose)₁ +hybrid/complex 3.2% (Man)₃(GlcNAc)₂ (Hex)₆ + (Man)₃(GlcNAc)₂ highmannose 1.1% (Hex)₇ + (Man)₃(GlcNAc)₂ high mannose 14.7% (Hex)₈ +(Man)₃(GlcNAc)₂ high mannose 0.6%

N-linked profile ASN³³³ for Glycovariant (600 μM CS) Relative GlycoformComposition Type abundance (Hex)₃ + (Man)₃(GlcNAc)₂ high mannose 0.1%(HexNAc)₁ (Deoxyhexose)₁ + hybrid/complex 0.9% (Man)₃(GlcNAc)₂ (Hex)₄ +(Man)₃(GlcNAc)₂ high mannose 0.5% (Hex)₁ (HexNAc)₂ (Deoxyhexose)₁ +hybrid/complex 1.3% (Man)₃(GlcNAc)₂ (Hex)₄ + (Man)₃(GlcNAc)₂ highmannose 6.2% (Hex)₂ (HexNAc)₂ (Deoxyhexose)₁ + hybrid/complex 0.3%(Man)₃(GlcNAc)₂ (Hex)₆ + (Man)₃(GlcNAc)₂ high mannose 2.3% (Hex)₇ +(Man)₃(GlcNAc)₂ high mannose 87.7% (Hex)₈ + (Man)₃(GlcNAc)₂ high mannose0.5% (Hex)₉ + (Man)₃(GlcNAc)₂ high mannose 0.1%

The above tables illustrate that samples treated with increasingconcentrations of castanospermine contain increasing amounts of highhexose, non-fucosylated glycoforms compared to the TRU-016 control. Athigher concentrations, the glycan distribution is almost fully shiftedto high hexose, non-fucosylated glycoforms, while at lowerconcentrations of CS a mixture of glycan types is observed, in adose-dependent fashion.

Example 19

Monosaccharide Analysis of TRU-016 Control and Glycovariants

This procedure was used to determine the monosaccharide content of theN-linked glycoforms displayed on TRU-016 control versus TRU-016glycovariant immunoglycoproteins. In this method, monosaccharides werereleased by hydrolysis then derivitized with a fluorescent modifier.Subsequent analysis of the labeled monosaccharides was accomplishedusing reverse phase chromatography coupled with fluorescence detection.Quantitation of the monosaccharides was accomplished by comparison oftest article monosaccharide peak areas to the peak areas obtained fromtitration of known concentrations of labeled monosaccharide standards.The monosaccharide content of the sample was then compared to the massof protein hydrolyzed and reported as the mole ratio of eachmonosaccharide per mole of the monomeric unit of the normally dimericTRU-016 protein.

Samples analyzed included the TRU-016 control and TRU-016 glycovariantproduced in the presence of 400 μM CS.

Monosaccharides were released from the protein by incubating 50 μg ofeach sample with 20% (v/v) trifluoroacetic acid at 100° C. for 4 hours.Following hydrolysis, the samples were dried down. Releasedmonosaccharides were resuspended in 200 μL of labeling mixturecontaining 30 mg/mL fluorescent derivative (2-AA, 2-Aminobenzoic Acid),20 mg/mL sodium cyanoborohydride, approximately 30 mg/mL sodium acetateand 15 mg/mL boric acid in methanol and incubated at 80° C. for 60minutes. The derivatization reaction was quenched by the addition of 200μL of mobile phase A [0.2% (v/v) n-butylamine, 0.5% (v/v) phosphoricacid, 1% (v/v) tetrahydrofuran]. A water blank was also hydrolyzed andderivitized to determine method specificity. Samples were then analyzedby reverse phase HPLC coupled with fluorescence detection. The quantityof monosaccharide in each sample was determined by comparison tostandard curves.

Representative chromatograms of the monosaccharide standards, TRU-016control and TRU-016 glycovariant (400 μM CS) are shown in FIG. 27. Asseen in the chromatograms, the mannose content of the TRU-016glycovariant was approximately double that of the TRU-016 control.GlcNAc content of the glycovariant decreased by about half from theTRU-016 control and fucose was absent in the TRU-016 glycovariantsample. In addition, glucose, which was below the limits of quantitationin the TRU-016 control, increased to 2-3 moles per mole of monomericprotein in the TRU-016 glycovariant sample. These results coupled withthe preceeding LCMS data are consistent with a shift from complex,predominantly G0F glycosylation on the TRU-016 control to a primarilyglucosylated high mannose form on the TRU-016 glycovariant.

It should be noted that TRU-016 glycosylation, as with allglycoproteins, is heterogeneous and there is potential for loss due tothe harsh conditions of this assay and resultant low recovery. Thus,these assays tend to underestimate the actual monosaccharide content ofa glycoprotein. In addition, all glycan species in the heterogeneousmixture contribute to the total monosaccharide content of a proteinsample, so the molar content of individual monosaccharides may notcorrelate directly to any single glycan species.

Example 20

Oligosaccharide Analysis of TRU-016 Control Versus TRU-016 Glycovariant

The purpose of this study was to characterize the oligosaccharidecontent of TRU-016 control and glycovariant test articles. In thismethod, oligosaccharides were first released from denatured protein byenzymatic digestion with PNGase-F. The released oligosaccharides werethen derivatized with a fluorescent label and separated by normal phasechromatography coupled with fluorescence detection. Each released glycanspecies will resolve to produce a characteristic oligosaccharide profileby which changes in glycosylation can be monitored.

Samples were initially reduced in a solution of 2% SDS and 1M betamercaptoethanol for 5 minutes at 100° C. Enzymatic release ofoligosaccharides was accomplished by incubation of samples with 2 μLPNGase-F at 37° C. for 3 hours. Following release, the oligosaccharideswere fluorescently labeled using the LudgerTag 2-AA glycan labeling kit(QA-Bio, LLC, Palm Desert, Calif.), following the manufacturer'srecommended procedures.

The glycans were purified from the excess labeling reagent using aQA-Bio S Cartridge following the procedures specified by themanufacturer. The labeled oligosaccharides were then separated by normalphase chromatography and monitored by fluorescence detection. Forfurther analysis of individual glycans, a separate analysis of TRU-016control was performed to collect fractions of the major glycan peaks forsubsequent analysis by MALDI-TOF.

Oligosaccharide profiles for the TRU-016 control and TRU-016glycovariant (400 μM CS) are shown in FIG. 28 with relative percent ofpeak area for the major glycan species indicated. These profiles areconsistent with LCMS and monosaccharide analysis data (alreadydescribed) as well as the known elution profile of oligosaccharidestandards. The major species in the control oligosaccharide profilecorrespond to the G0F, G1F and G2F glycans standards (not shown).Typically, these, fucosylated, complex oligosaccharide standards elutebetween 18.5 and 23 minutes, while non-fucosylated, high mannoseoligosaccharides elute between 24.5 and 30 minutes with the conditionsused for this analysis. Thus, the TRU-016 control oligosaccharidesexhibit a profile consistent with fucosylated, complex glycans with onlya very minor contribution from oligo-mannose glycans. The TRU-016glycovariant, however, demonstrates a strong shift to high mannoseoligosaccharides, with only a minor, residual contribution fromfucosylated, complex glycans.

In addition to the analytical comparison of oligosaccharide profiles, apreparative analysis of TRU-016 control was performed and the majorglycan species collected for further analysis. The two major glycans ofTRU-016 were identified by this method:

-   -   Most abundant species (˜20 minutes RT):        (HexNAc)₄(Hex)₃(DeoxyHex)₁    -   Secondary species (˜21.5 minutes RT): (HexNAc)₄(Hex)₄(DeoxyHex)₁    -   These structures correspond to the G0F and G1F glycoforms.        The results of the oligosaccharide analysis of TRU-016        glycovariant samples are consistent with the LCMS and        monosaccharide analyses and with a shift from complex,        fucosylated glycoforms to non-fucosylated, glucosylated, high        mannose forms.        Analytical Summary

Taken together, the LCMS, monosaccharide and oligosaccharide analysesdemonstrate that production of TRU-016 in the presence of a sufficientamount of castanospermine alters the N-linked oligosaccharide contentfrom primarily fucosylated, complex type glycans (G0F and G1F) to afamily of non-fucosylated, glucosylated, high hexose glycans dominatedby a (Hex)₇+(Man)₃(GlcNAc)₂ glycoform. Monosaccharide analysis of theglycovariant demonstrates the presence of 2 or more moles of glucose permole of monomeric protein. Because there is only one N-linkedglycosylation site per monomeric unit of TRU-016, this data indicatesthat the composition of the major N-linked glycan species, generated byproduction of TRU-016 in the presence of castanospermine, is(Glc)₃(Man)₄+(Man)₃(GlcNAc)₂ and/or (Glc)₂(Man)₅+(Man)₃(GlcNAc)₂.

Monitoring the CD16 binding/ADCC activity of the glycovariant as afunction of CS concentration in the cell culture allows identificationof the CS concentration at which these functional activities becomemaximal. In this same manner, LCMS analysis of glycoform composition asa function of CS concentration allows monitoring of the shift incomposition as it relates to CS concentration and, hence, to CD16binding/ADCC enhancement. For example, production of TRU-016 by CHO-K1cells in the presence of 50 μM CS (Table 9) yields a glycovariantproduct that has maximal ADCC activity while the glycan composition hasshifted to ˜50% (Hex)₇+(Man)₃(GlcNAc)₂. This indicates that a completeshift to a single glycoform is not necessary to achieve the observedincrease in activity. This data further suggests that the glycoformsattached to the two N-linked sites in dimeric SMIP molecules need not beidentical or need not both consist of the non-fucosylated, glucosylated,high mannose form to achieve optimal CD16 binding or ADCC. Modificationof only one of the SMIP N-linked oligosaccharides to thenon-fucosylated, glucosylated, high mannose type may be sufficient tomaximize the CD16 binding and ADCC properties of the SMIP.

While the compositions and methods of this invention have been describedin terms of the above-described exemplary embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. More specifically, it will beapparent that certain agents which are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

The references cited herein throughout, to the extent that they provideexemplary details supplementary to those set forth herein, are allspecifically incorporated herein by reference.

1. A composition comprising recombinant immunoglycoprotein molecules,wherein the recombinant immunoglycoprotein molecules each comprise anN-linked glycosylation site for linkage of oligosaccharide, and whereinat least 60% of the N-linked oligosaccharides of the N-linkedglycosylation site have a hexose content of at least ten hexosemolecules.
 2. The composition of claim 1, wherein the immunoglycoproteinmolecules exhibit at least 5-fold higher ADCC compared to controlimmunoglycoprotein molecules of the same encoded amino acid sequenceproduced in CHO-K1 cells in the absence of a carbohydrate modifier. 3.The composition of claim 1, wherein at least 60% of the N-linkedoligosaccharides of the N-linked glycosylation site contain no fucose.4. The composition of claim 1 formulated in a pharmaceuticallyacceptable carrier or diluent.
 5. The composition of claim 4, whereinthe composition is sterile.
 6. The composition of claim 1 comprising atleast 100 g of immunoglycoprotein molecules and a pharmaceuticallyacceptable carrier or diluent.
 7. The composition of claim 1 comprisingat least 100 liters of culture medium.
 8. The composition of any one ofclaims 1-3, wherein the recombinant immunoglycoprotein molecules arebispecific in antigen-binding.
 9. The composition of any one of claims1-3, wherein the recombinant immunoglycoprotein molecules aretrispecific in antigen-binding.
 10. The composition of any one of claims1-3, wherein the recombinant immunoglycoprotein molecule is an antibodyor a small modular immunopharmaceutical.
 11. The composition of claim10, wherein the small modular immunopharmaceutical comprises amino acids24-496 of SEQ ID NO:2.